Assay cartridges and methods of using the same

ABSTRACT

Assay modules, preferably assay cartridges, are described as are reader apparatuses which may be used to control aspects of module operation. The modules preferably comprise a detection chamber with integrated electrodes that may be used for carrying out electrode induced luminescence measurements. Methods are described for immobilizing assay reagents in a controlled fashion on these electrodes and other surfaces. Assay modules and cartridges are also described that have a detection chamber, preferably having integrated electrodes, and other fluidic components which may include sample chambers, waste chambers, conduits, vents, bubble traps, reagent chambers, dry reagent pill zones and the like. In certain preferred embodiments, these modules are adapted to receive and analyze a sample collected on an applicator stick.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. patent application Ser. No. 10/744,726,filed Dec. 23, 2002, which claims priority of U.S. ProvisionalApplication No. 60/436,569, filed Dec. 26, 2002, which is incorporatedherein by reference.

FIELD OF THE INVENTION

This application relates to apparatuses, systems, kits and methods forconducting chemical, biochemical and/or biological assays on a sample.These apparatuses include assay cartridges and cartridge readers forconducting these assays. The application also describes electrode arraysfor use in assays, methods of preparing and using these electrode arraysand diagnostic devices comprising the arrays. These electrode arrays maybe incorporated into the cartridges and apparatuses of the invention.

BACKGROUND OF THE INVENTION

Clinical measurements have been traditionally carried out in centralclinical labs using large clinical analyzers that can handle largenumbers of samples in batch mode. These laboratories are staffed bytrained personnel that are capable of maintaining and running thesecomplex analyzers. There is a growing desire to move clinicalmeasurements from the central lab to the “point of care”, e.g., theemergency room, hospital bedside, physicians office, home, etc. Point ofcare measurements allow a care provider or patient to quickly makedecisions based on diagnostic information, as opposed to having to waithours or days to receive laboratory results from a clinical lab. Thedifficulty in developing point of care diagnostic systems has beenmaking them small enough and easy enough to use so that they can be usedby unskilled operators in decentralized clinical settings, but at thesame time maintaining the low cost, diverse assay menu, and/or highperformance of tests carried out on traditional clinical analyzers incentral laboratories.

SUMMARY OF THE INVENTION

The invention relates in part to assay modules, preferably assaycartridges. An assay module of the invention incorporates one or morefluidic components such as compartments, wells, chambers, fluidicconduits, fluid ports/vents, valves, and the like and/or one or moredetection components such as electrodes, electrode contacts, sensors(e.g. electrochemical sensors, fluid sensors, mass sensors, opticalsensors, capacitive sensors, impedance sensors, optical waveguides,etc.), detection windows (e.g. windows configured to allow opticalmeasurements on samples in the cartridge such as measurements ofabsorbance, light scattering, light refraction, light reflection,fluorescence, phosphorescence, chemiluminescence,electrochemiluminescence, etc.), and the like. A module may alsocomprise reagents for carrying out an assay such as binding reagents,detectable labels, sample processing reagents, wash solutions, buffers,etc. The reagents may be present in liquid form, solid form and/orimmobilized on the surface of solid phase supports present in thecartridge. In certain embodiments of the invention, the modules includeall the components necessary for carrying out an assay. In otherembodiments, the invention also includes a module reader adapted toreceive the module and carry out certain operations on the module suchas controlling fluid movement, supplying power, conducting physicalmeasurements on the cartridge, and the like.

The invention also relates, in part, to a method of performing aplurality of assays wherein an assay dependent signal is measured usinga plurality of electrodes. Preferably, at least one of the electrodes isused as a working electrode for measuring an assay dependent signal and,subsequently, as a counter electrode for measuring a different assaydependent signal at a different electrode. In one preferred embodiment,at least two of the electrodes are used as a working electrode and,subsequently, as a counter electrode. Most preferably, the method usesat least a dedicated counter electrode, a dedicated working electrodeand two or more additional electrodes, each of which is used as aworking electrode for measuring an assay dependent signal and,subsequently, as a counter electrode for measuring a different assaydependent signal at a different electrode.

In another preferred embodiment, a method of performing a plurality ofbiochemical assays using a plurality of electrodes is disclosed. Themethod comprises the steps of applying electrical energy between firstand second electrodes, measuring an assay dependent signal at the secondelectrode, applying electrical energy between the second electrode and athird electrode and measuring an assay dependent signal at the thirdelectrode. The measured assay dependent signal is, preferably, selectedfrom electrical current, electrical potential and/or electrode-inducedluminescence. The second and third electrodes can each have an assayreagent immobilized thereon. Furthermore, each electrode can have adifferent assay reagent immobilized thereon where each assay reagent canbe specific for a different analyte of interest.

In one embodiment, the plurality of electrodes can be arranged within aflow cell. In a preferred embodiment, the flow cell can have a flow cellpath along which the electrodes may be arranged. The electrodes can bearranged along the path, sequentially. Moreover, the electrodes can bearranged such that the first electrode is adjacent the second electrodeand the second electrode is adjacent the third electrode. The electrodescan be arranged within a single detection chamber. Additionally, theelectrodes may comprise printed carbon ink. Further, the assay reagentsmay be immobilized on the electrode surface within an assay domaindefined by a dielectric layer on the electrodes.

In yet another embodiment, the electrodes may have electrical leads forsupplying electrical energy to the electrodes. The electrical leads maycomprise exposed surfaces that at least partially define an inletconduit in fluid communication with the flow cell. The method may theninclude the further step of applying an inlet conduit interrogationpotential between the exposed surfaces of the electrical leads todetermine the presence or composition of fluid in the inlet conduit.Preferably the interrogation potential would be of insufficientmagnitude to induce electrochemiluminescence.

According to another aspect of the invention, an apparatus forperforming a plurality of biochemical assays is disclosed. The apparatusmay comprise a plurality of electrodes comprising at least one dedicatedworking electrode, at least one dual-role electrode and at least onededicated counter electrode. The dedicated working and dual-roleelectrodes preferably have deposited thereon an assay reagent. Thedual-role electrode is advantageously configured to operate first as theworking electrode and then as the counter electrode. The assay reagentis preferably a binding reagent that is specific for an analyte ofinterest and may also be different for each of the dedicated working anddual-role electrodes.

Still further, the plurality of electrodes may be arranged within a flowcell, along the flow cell path. Preferably, the dedicated counterelectrode is adjacent the dual-role electrode and the dual-roleelectrode is adjacent the dedicated working electrode. In addition, theplurality of electrodes are preferably arranged within a singledetection chamber. The plurality of electrodes may comprise printedcarbon ink. The dedicated working and dual-role electrodes may haveassay reagents immobilized thereon within an assay domain defined by adielectric layer.

The dedicated working, dual-role and dedicated counter electrodespreferably have corresponding electrical leads for supplying electricalenergy to the electrodes. Preferably, at least two non-adjacentelectrical leads would have an exposed surface located thereon. Theseexposed surfaces of the electrical leads preferably at least partiallydefine an inlet conduit in fluid communication with a flow cell so thatfluid present within the inlet conduit is in electrical contact with theexposed surfaces. In such a preferred embodiment, the exposed surfacesmay be configured to apply an inlet conduit interrogation potentialbetween exposed surfaces to determine the presence or composition offluid in the inlet conduit. Additionally, the apparatus is preferablyconfigured such that the applied interrogation potential between exposedsurfaces is of insufficient magnitude to induce electrochemiluminescenceat the corresponding electrodes.

In yet another embodiment, the apparatus can be configured with anoptical detector for detecting luminescence generated at the dedicatedworking and dual-role electrodes. Alternatively, the apparatus maycomprise a voltmeter for measuring potentials at the dedicated workingand dual-role electrodes. In yet another alternative embodiment, theapparatus may comprise an ammeter for measuring electrical current atsaid dedicated working and dual-role electrodes. Preferably, theelectrodes are housed in a disposable assay cartridge and the opticaldetector(s), voltmeter(s), and/or ammeter(s) are housed in a separatere-usable cartridge reader.

In accordance with another aspect of the invention, a cartridge forconducting a plurality of assays may comprise a flow cell having aninlet, outlet and a detection chamber. The detection chamber preferablycomprises a plurality of electrodes arranged in a one dimensional arraywherein at least a first electrode has a first assay reagent immobilizedthereon. According to certain preferred embodiments, the electrodes maycomprise carbon ink. The electrodes preferably have a plurality ofelectrical leads that supply electrical energy to the electrodes. Inaddition, the cartridge may comprise a second electrode arrangedadjacent to the first electrode, the second electrode preferably havinga second assay reagent immobilized thereon.

According to one embodiment, the cartridge preferably has a detectionchamber with at least one detection chamber surface. Preferably, atleast a portion of the detection chamber surface would be transparent.Still further, the cartridge may comprise an optical detector adaptedand arranged to detect luminescence from the detection chamber.Preferably, the optical detector is provided in a separate cartridgereader.

In accordance with another aspect of the invention, a method isdisclosed for conducting an electrochemiluminescence measurement whereinimpedance is measured between two electrodes and whereinelectrochemiluminescence is induced at one of the two electrodes. Theimpedance is measured between the two electrodes in a measurementchamber to detect the presence of air bubbles. The impedance measurementstep is preferably conducted using electrical energy that isinsufficient for generating electrochemiluminescence at the electrodes.Additionally, the impedance measurement may be conducted using either aDC impedance measurement or, more preferably, an AC impedancemeasurement.

According to yet another aspect of the invention, a method of depositingassay reagents on an electrode surface, preferably comprising carbonink, to form an assay domain is disclosed. The method comprises thesteps of dispensing a predetermined volume of the assay reagents on theelectrode surface using impact-driven fluid spreading to coat apredefined region having a predefined assay reagent area on theelectrode surface. The predetermined volume of said assay reagents ispreferably dispensed at a velocity greater than 200 centimeter persecond (cm/s). Preferably the predefined assay reagent area is largerthan the steady-state spreading area of the predetermined volume of theassay reagents on the electrode surface. More preferably the predefinedassay reagent area is at least twice the steady-state spreading area ofthe predetermined volume of the assay reagents on the electrode surface.The method would preferably use a fluid dispenser utilizing using afluid micro-dispenser such as a micro-pipette, micro-syringe, solenoidvalve dispenser, piezo-driven dispenser, ink-jet printer, bubble jetprinter, etc. Also, the assay reagents are preferably substantially freefrom surfactants.

According to one embodiment, the electrode surface preferably comprisesa material having advancing and retreating contact angles for the assayreagents (preferably, aqueous solutions having contact angles thatapproximate that of water) that differ. More preferably, this differenceis at least 10 degrees. The electrode surface need not be plasmatreated. Additionally, the predefined region is preferably defined by adielectric material having dielectric advancing and retreating contactangles for the assay reagents. The dielectric retreating contact angleis preferably greater than the electrode surface retreating contactangle. More preferably, the dielectric advancing and retreating contactangles are about equal to each other but greater (preferably, by morethan 10 degrees) than the electrode surface retreating contact angle.Most preferably, the dielectric advancing and retreating contact anglesare within about 20 degrees of each other. Also, the predeterminedvolume may preferably be selected such that any assay reagents thatspread onto the dielectric material retreat to an interface between thedielectric material and the electrode surface that defines thepredefined region.

A further aspect of the invention relates to a method of adsorbing assayreagents on a carbon ink electrode. The method may include the steps ofwashing the electrode and then treating the electrode with solutioncontaining the assay reagents. The washing step preferably employs awashing solution comprising a surfactant; e.g., a non-ionic surfactantselected from the surfactants known by the trade names of Brij, Triton,Tween, Thesit, Lubrol, Genapol, Pluronic (e.g., F108), Tetronic,Tergitol, and Span, most preferably Triton X100. Additionally, after thewashing step and prior to the treating step, the electrode may be rinsedwith a surfactant free solution. Preferably, the electrode is soaked inthe surfactant free solution for about one hour.

In accordance with a still further aspect of the invention, a method offorming an assay domain comprising an assay reagent is disclosed.Preferably, in accordance with such method, a predefined region of asurface is treated with an avidin solution so as to form an adsorbedavidin layer within the predefined region of the surface. Next, theadsorbed avidin layer is preferably treated with a solution comprisingthe assay reagent, the assay reagent being linked to biotin. Morepreferably, the avidin solution is dried on the surface prior totreatment with the assay reagent solution. The method may also employthe step of washing the adsorbed avidin layer prior to treatment withthe assay reagent solution. The surface may be a carbon ink electrode.The predefined region is preferably defined by a boundary adapted toconfine the avidin and/or assay reagent solutions to the predefinedregion (most preferably both solutions are confined to the pre-definedregion). The boundary can be defined by a dielectric layer.

According to another aspect of the invention, a method of forming aplurality of assay domains is disclosed wherein one of a plurality ofpredefined regions of a surface are treated with an avidin solution soas to form an adsorbed avidin layer within the predefined region of thesurface. The adsorbed avidin layer is then preferably treated with asolution comprising an assay reagent linked to biotin. These steps maythen be repeated for each of the plurality of assay domains. Morepreferably, the avidin solution is dried on the surface prior totreatment with the assay reagent solution. The method may also employthe step of washing the adsorbed avidin layer prior to treatment withthe assay reagent solution. The surface may be a carbon ink electrode.The predefined region is preferably defined by a boundary adapted toconfine the avidin and/or assay reagent solutions to the predefinedregion (most preferably both solutions are confined to the pre-definedregion). The boundary can be defined by a dielectric layer.

The assay reagent in each domain may be the same or may be different.Assay reagents that may be used include, but are not limited to,antibodies, fragments of antibodies, proteins, enzymes, enzymesubstrates, inhibitors, cofactors, antigens, haptens, lipoproteins,liposaccharides, cells, sub-cellular components, cell receptors,membrane fragments, viruses, nucleic acids, antigens, lipids,glycoproteins, carbohydrates, peptides, amino acids, hormones,protein-binding ligands, pharmacological agents, membrane vesicles,lipsomes, organelles, bacteria or combinations thereof. Preferably, theassay reagents are binding reagents capable of specifically binding toan analyte of interest or, alternatively, of competing with an analyteof interest for binding to a binding partner of the analyte of interest.Especially preferred assay reagents are antibodies and nucleic acids.

According to one embodiment, the avidin solution for forming one, or aplurality, of assay domains may comprise a polymeric form of avidin. Thepolymeric form of avidin may be formed by forming a solution of avidinand a cross-linking molecule, the cross-linking molecule preferablyhaving a plurality of biotin groups. The ratio of the cross-linkingmolecule to avidin is preferably between 0.01 and 0.25. The method offorming an assay domain can preferably include the step of washing theassay domain or plurality of assay domains. More preferably, the washsolution comprises blocking agent, wherein the blocking agent can be aprotein or biotin.

The invention also relates to assay cartridges employing the electrodearrays and/or binding domains employing these electrode described above(and adapted for carrying out the methods described above for usingthese arrays and domains) and assay cartridge readers for operating andanalyzing these cartridges. The invention also relates to assay systemscomprising these cartridges and cartridge readers. The cartridges andreaders, preferably, comprise the necessary fluidics and control systemsfor moving sample and reagent fluids, collecting waste fluids, removingand/or introducing bubbles from liquid reagents and/or samples,conducing physical measurements on the samples and/or extractingsamples.

The invention also relates to assays cartridges comprising a samplechamber preferably having a sealable closure, an optional waste chamberand a detection chamber (preferably, a detection chamber having one ormore binding domains having immobilized binding reagents, morepreferably, one or more binding domains on one or more electrodes, mostpreferably an electrode array of the invention as described above). Thedetection chamber is connected to the sample chamber via a sampleconduit and, if present, to the waste chamber via a waste conduit. Theassay cartridge may also include a sample chamber vent port connectedthe sample chamber and/or a waste chamber vent port connected to thewaste chamber. The sample can include a capillary break, preferably az-transition. The z-transition preferably includes a fluid conduitsegment that connects two planar fluidic networks of the cartridge. Thecapillary break may alternatively comprise a double z-transition.

In another embodiment of an assay cartridge that includes: a ventedsample chamber with an introduction port and a sealable closure; avented waste chamber; and a detection chamber (preferably, a detectionchamber having one or more binding domains having immobilized bindingreagents, more preferably, one or more binding domains on one or moreelectrodes, most preferably an electrode array of the invention asdescribed above) connected to the sample and waste chambers via sampleand waste conduits, respectively, one or more fluidic networks may bedefined within the cartridge's body by one or more cover layers mated toa side of the cartridge body. A second cover layer, or set of coverlayers, may be mated to a second side of the cartridge body to form oneor more additional second side fluidic networks therebetween, the firstand second side fluidic networks being in fluidic communication by atleast one though-hole within the cartridge body. The fluidic networksmay be defined, at least in part, by recesses in the cartridge bodyand/or cover layers. In addition, at least one of the fluidic networksmay be defined, at least in part, by apertures within a gasket layerdisposed between the cartridge body and at least one cover layer.

Additionally, embodiments including a z-transition capillary break, thez-transition may comprise, in series, first, second, third, fourth andfifth sample conduit segments, each of the segments being connected atan angle to the adjacent segments and the segments being oriented sothat the first and fifth segments are in the first fluidic networks, thethird segment is in the second fluidic network and the second and fourthsegments are cartridge body through-holes.

Still further, the assay cartridge may comprise a dry reagent in thesample conduit. The dry reagent may comprise, e.g., a labeled bindingreagent, a blocking agent, an ECL coreactant and/or an extraction bufferneutralization reagent. In yet another embodiment, the assay cartridgemay comprise an air vent port connected to the sample conduit. In stillyet another embodiment, the assay cartridge may comprise a ventedreagent chamber and a reagent chamber conduit connecting the reagentchamber with the sample conduit. The reagent chamber may comprise aliquid reagent which may optionally be contained within a reagentampoule in the reagent chamber. The reagent chamber conduit may also beconnected to an air vent port.

The reagent conduit may include a dry reagent; the dry reagent maycomprise, e.g., a labeled binding reagent, a blocking agent, an ECLcoreactant and/or an extraction buffer neutralization reagent. Theliquid reagent may be, e.g., a wash buffer, an extraction buffer, anassay diluent and/or an ECL read buffer. The extraction buffer is,preferably, nitrous acid or a nitrate salt.

In another embodiment the assay cartridge may further comprise a secondreagent chamber holding a second liquid reagent, a second reagentchamber vent port connected to the second reagent chamber and a secondreagent chamber conduit connecting the second reagent chamber with thesample conduit.

The detection chambers in the cartridges of the invention preferablyinclude an array of binding reagents as described above. Still further,the detection chamber may comprise one or more electrodes having bindingreagents immobilized thereon as described above.

In other embodiments the assay cartridge may further comprise a secondwaste chamber, a second waste chamber vent port connected to the secondwaste chamber and a second detection chamber connected to the samplechamber or the first sample conduit by a second sample conduit and tothe second waste chamber by a second waste conduit. In addition, atleast a portion of one wall of the detection chamber may besubstantially transparent to allow optical monitoring of materials inthe detection chamber. The assay cartridge may also comprise a seconddetection chamber connected to the sample chamber or the first sampleconduit by a second sample conduit and to the first waste chamber by asecond waste conduit. Similarly, at least a portion of one of the coverlayers may be substantially transparent to allow the monitoring of fluidflow within said cartridge.

In other embodiments, the cover layers may have a first regioncomprising a patterned array of immobilized binding reagents defining asurface of the detection chamber and a second region having a dryreagent thereon defining a surface of the sample conduit. The cartridgemay also have two second side cover layers defining two second sidefluidic networks and a first side bridge cover layer that connects thetwo second side fluidic networks. In certain embodiments, the dryreagents may be on the first side bridge cover layer.

In yet a still further embodiment, an assay cartridge for analyzing asample collected with an applicator stick comprising a shaft and asample collection head, may comprise a sample chamber having anelongated cavity that has a first elongated region and a secondelongated region, the regions being oriented at an angle with respect toeach other to bend the shaft upon insertion of the applicator stick intothe sample chamber and promote fracture of the shaft. The angle ispreferably between 30 and 70 degrees. Also, in some embodiments thecross-sectional area of the cavity is less than 2 times the width of theapplicator stick head. The fracture preferably produces a shortenedstick fragment that includes the sample collection head where the lengthof the fragment is less than the length of the cavity. The cartridgealso may include a sealable closure for sealing the sample compartmentwith the shortened stick fragment in the cavity.

Other embodiments for an assay cartridge may comprise an extractionreagent chamber for holding an extraction reagent, a sample chamberhaving sample introduction port with a sealable closure wherein thesample chamber is adapted to receive an applicator stick and a firstdetection chamber (preferably, a detection chamber having one or morebinding domains having immobilized binding reagents, more preferably,one or more binding domains on one or more electrodes, most preferablyan electrode array of the invention as described above) connected to thesample chamber by a first sample conduit. The sample chamber isconnected to the extraction reagent chamber by an extraction reagentchamber conduit. A filter may optionally be included between the samplechamber and the sample conduit. The sample and extraction reagentconduits may be connected to and arranged along the length of thecavity. The extraction reagent, preferably, comprises nitrous acid or anitrate salt.

Yet another embodiment of an assay cartridge comprises a wash reagentchamber for holding a wash reagent and a detection chamber (preferably,a detection chamber having one or more binding domains havingimmobilized binding reagents, more preferably, one or more bindingdomains on one or more electrodes, most preferably an electrode array ofthe invention as described above), wherein the wash reagent chamber andthe waste chamber are connected to the detection chamber via a washconduit and a waste conduit, respectively. Alternatively, the wastechamber may be connected to the detection chamber via a waste conduitand the wash reagent chamber connected to the sample conduit via a washconduit.

In accordance with another aspect of the invention, a method ofperforming a cartridge based assay is disclosed. The method generallycomprises moving the sample from the sample chamber into the firstsample conduit branch. The dry reagent is reconstituted in the sampleand a sample slug having a predetermined volume is moved into thedetection chamber and then into the waste chamber. Reagent is then movedinto the detection chamber and a signal is measured.

The step of moving the sample into the sample conduit may involveopening the sample vent port and applying a vacuum to the first wastechamber vent port. The sample slug may be moved into the detectionchamber by opening the air vent port and applying a vacuum to the firstwaste chamber vent port. Moving the reagent may be accomplished byopening the reagent vent port and applying vacuum to the first wastechamber vent port. Optionally, moving the reagent may also compriseopening the air vent port to segment the reagent.

The assay may be a binding assay where the detection chamber comprisesone or more immobilized binding reagents and the first dry reagentcomprises one or more labeled binding reagents. The signal may be anelectrochemiluminescent signal wherein the detection chamber furthercomprises electrodes, the one or more labeled binding reagents cancomprise one or more electrochemiluminescent labels and the firstreagent may comprise an electrochemiluminescence coreactant.

In certain embodiments the dry reagent may be reconstituted by movingthe sample back and forth over the dry reagent. In addition, the slug ofsample may be moved back and forth in the detection chamber. Movingfluids back and forth can be accomplished by opening the air or samplechamber vent port and alternating between applying positive and negativepressure at the waste chamber vent port.

Selective control of fluid movement may be attained by moving sampleand/or reagent for predetermined periods of time. Alternatively, someembodiments may move sample and/or reagent until the sample and/orreagent reach predetermined locations. In addition, certain embodimentsmay use fluid sensors to determine when the sample and/or reagent reachthe predetermined locations. The slug of sample may be mixed in thedetection chamber by moving the slug back and forth within the detectionchamber. In certain embodiments the sample conduit and/or reagentconduit may comprise a z-transition that act as a capillary break.

The method may also comprise adding the sample to the sample chamberthrough a sample introduction port and sealing the sample introductionport. The invention includes embodiments where the sample is a liquidsample and/or the sample contains a solid matrix. The method may also beutilized where the sample chamber is connected to the sample chambervent through an extraction chamber containing an extraction reagent.

In yet another embodiment, the cartridge based assay method may becarried out on a cartridge having a second vented waste chamber and asecond detection chamber connected to the sample chamber by a secondsample conduit branch containing a second dry reagent and to the secondwaste chamber by a second waste conduit. The method would furthercomprise moving the sample from the sample chamber into the secondsample conduit branch, reconstituting the second dry reagent in thesample, moving a second slug of sample having a predetermined volumeinto the second detection chamber, moving the second slug in the seconddetection chamber into the second waste chamber, moving reagent into thesecond detection chamber and measuring a signal from the seconddetection chamber. The reagent conduit may also comprise a third dryreagent. Other embodiments may employ a second reagent chambercontaining a second reagent, wherein the second reagent chamber isconnected to the sample conduit or the first reagent conduit through asecond reagent conduit and the second reagent is moved into thedetection chamber.

Still other embodiments of a method for performing a cartridge basedassay may comprise the steps of moving the sample from the samplechamber into the first sample conduit, reconstituting the first dryreagent in the sample, moving a slug of the sample into the firstdetection chamber, moving the sample in the first detection chamber intothe waste chamber, moving the reagent into the detection chamber andmeasuring a signal from the detection chamber. Such a method may utilizea cartridge having a detection chamber that has an elongated dimensionwhere the sample and reagent conduits connect to the detection chamberat substantially opposite ends of the detection along the elongateddimension. Additionally, the method may be performed such that thesample slug moves through the detection chamber along a path in aforward direction and the reagent moves through the detection chamberalong the path in the reverse direction.

In still further embodiments, the method may be performed on a cartridgehaving second waste and detection chambers where the second detectionchamber is connected to the first detection chamber conduit by a secondreagent chamber conduit and to the second waste chamber by a secondwaste conduit. The method may include the step of moving the reagentinto the second detection chamber and measuring a signal from the seconddetection chamber.

In accordance with another aspect of the invention, a method forpreparing a sample for analysis may include the steps of inserting anapplicator stick, which has a shaft and a sample collection head, usedto collect a sample into a cartridge having a sample chamber, breakingthe shaft of the applicator stick into a shaft segment and a headsegment and sealing the head segment in the sample chamber. Theinserting step may occur concurrently with the breaking step or mayoccur prior to the breaking step. The breaking step may be carried outby applying a force perpendicular to the shaft. Optionally, the samplechamber may include force focusing elements.

In yet other embodiments, the assay cartridge used in the method forpreparing a sample for analysis may have a sample chamber that has anelongated cavity, the elongated cavity comprising a first elongatedregion and a second elongated region wherein the two regions areoriented at an angle with respect to each other. The inserting step of amethod using such an assay cartridge may comprise pushing the samplecollection head through the first region and into the second regioncausing the shaft to bend and break. In certain embodiments, theapplicator stick breaks at a predefined weak point located on shaft.Preferably, the weak point is located between the first and secondregions when the applicator stick is fully inserted.

In a still further embodiment, the method of preparing a sample foranalysis may comprise passing an extraction reagent through the samplechamber having the head segment to form a sample liquid and thenintroducing the sample liquid into the detection chamber. In addition,the sample conduit connected to the sample chamber may comprise afilter. Still further, the cartridge may have a bubble trap chamberconnected to the sample chamber and the method may further include thestep of introducing the sample liquid into the bubble trap and removingbubbles from the sample liquid prior to introducing the sample liquidinto the detection chamber.

In certain embodiments the bubble trap chamber may connect to the samplechamber via a bubble trap conduit that is connected to the sampleconduit wherein the bubble trap conduit is connected to the bubble trapchamber at or near the bottom of the bubble trap chamber. In such anembodiment, the step of removing bubbles may comprise maintaining thesample liquid in the bubble trap for a sufficient amount of time toallow any bubbles that might be present in the sample liquid to rise tothe top of the sample liquid allowing a reduced bubble portion of thesample liquid to then be removed from the bubble trap chamber throughthe bubble trap chamber conduit. Alternatively, the bubble trap chambermay be interposed between the sample conduit and the detection chamberand may have an inlet connected to the sample conduit and an outletconnected to the detection chamber wherein the outlet is arranged at ornear the bottom of the bubble trap chamber. In such an alternativeembodiment, the step of removing bubbles may comprise maintaining thesample liquid in the bubble trap for a sufficient amount of time toallow any bubbles that might be present in the sample liquid to rise tothe top of the sample liquid allowing a reduced bubble portion of thesample liquid to be removed from the bubble trap chamber through thebubble trap chamber conduit.

In accordance with yet another aspect invention, an assay system maycomprise an assay cartridge in accordance with any of the embodiments ofthe present invention and a cartridge reader adapted to carry out anassay using the cartridge.

Additionally, a kit is disclosed that may comprise an assay cartridge inaccordance with any of the embodiments of the present invention and anapplicator stick. The applicator stick of such a kit may have apredefined weak point.

The invention also relates to cartridge readers adapted to control andcarryout measurements using the above described cartridges, systemscomprising the above described cartridges and a cartridge reader andkits including the cartridge and one or more reagents and/or applicatorsticks used in assays carried out employing the cartridges.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a depicts a simplified pictorial representation of acartridge-based assay module.

FIG. 1 b depicts one embodiment of an assay cartridge having twodetection chambers and two banks of individually addressable electrodes.

FIG. 1 c illustrates an exploded assembly of one embodiment of anelectrode array.

FIG. 2 is a pictorial representation of an electrode array havingmatched electrical lead resistances.

FIGS. 3 a-3 e illustrate various configurations of an electrodes arrayfor use with a pair-wise firing schemes.

FIGS. 3 f-3 g illustrate two possible configurations of an electrodearray employing a single, common counter electrode.

FIG. 4 depicts the electrode array of FIG. 3 a in one embodiment of anassay cartridge.

FIG. 5 is an image of electrochemiluminescence emitted from an electrodearray where one of the electrodes has an air bubble on the electrodesurface.

FIGS. 6 a and 6 b are images of electrochemiluminescence from electrodearrays that are untreated (FIG. 6 a) or that have been pre-washed with asurfactant (FIG. 6 b).

FIG. 7 a illustrates the use of a localized washing apparatus havingconcentric tubes.

FIG. 7 b is a cross-sectional view of the localized washing apparatusdepicted in FIG. 7 a.

FIG. 8 plots the contact angle of drops of fluid on carbon ink anddielectric ink surfaces as a function of the dispensing velocity.

FIG. 9 is a schematic representation of one embodiment of an assaycartridge illustrating various fluidic components.

FIG. 10 depicts the fluidic network in accordance with the schematicrepresentation of FIG. 9.

FIGS. 11 a-11 c are top, bottom and isometric views, respectively, ofthe assay cartridge of FIG. 9; FIG. 11 a illustrates the fluidicnetworks formed on one side of the cartridge, FIG. 11 b illustrates thefluidic network formed on the other side of the cartridge and FIG. 11 cprovides an isometric view with phantom lines to illustrate the entirecartridge fluidic network as seen within the cartridge body.

FIG. 12 is a bottom view of the assay cartridge of FIG. 9 illustratingone preferred layout for fluidic detectors to detect/monitor fluidmovement.

FIG. 13 a is an exploded assembly drawing illustrating the laminarassemblage for the assay cartridge depicted in FIG. 9.

FIG. 13 b is a detail drawing of the gasket and electrode array coverlayer depicted in FIG. 13 a.

FIG. 14 a is a schematic representation of another embodiment of anassay cartridge illustrating various fluidic components.

FIG. 14 b is an exploded assembly drawing illustrating the laminarassemblage for the two-piece assay cartridge depicted in FIG. 14 a.

FIG. 14 c is a detail drawing of the gasket and electrode array coverlayer depicted in FIG. 14 b.

FIG. 15 a is a top view of the upper cartridge component of the assaycartridge depicted in FIG. 14 b.

FIGS. 16 a and 16 b are top and bottom views, respectively, of the lowercartridge component of the assay cartridge depicted in FIG. 14 b.

FIG. 17 is a bottom view of the assay cartridge of FIG. 14 billustrating one preferred layout for fluidic detectors todetect/monitor fluid movement.

FIGS. 18 a and 18 b are top and bottom isometric views, respectively,depicting the fluidic network in accordance with the schematicrepresentation of FIG. 14 a.

FIG. 19 is a bottom view of the upper cartridge component of the assaycartridge depicted in FIG. 14 b illustrating one embodiment of integralfilters.

FIG. 20 is a bottom isometric view of an alternative assay cartridgeembodiment illustrating filter inserts.

FIG. 21 is an isometric view of the assay cartridge depicted in FIG. 14b having assay reagent ampoules inserted therein, illustrating oneembodiment for an assay reagent release mechanism.

FIG. 22 illustrates one embodiment for a drop-in assay reagent blisterpack assembly and integrated assay reagent release (piercing) mechanism.

FIG. 23 illustrates one embodiment for a cartridge reader thatincorporates various subsystems for performing a predetermined assay.The cartridge reader is depicted holding one embodiment of an assaycartridge.

FIG. 24 illustrates one preferred valve configuration for the assaycartridge depicted in FIG. 14 a.

FIG. 25 is the schematic representation shown in FIG. 14 a depicting thearrangement of fluidic components and locations of fluid detectors.

FIGS. 26 a through 26 c illustrate one preferred manner of operating theassay cartridge depicted in FIG. 25.

FIG. 27 is a cross-sectional view of a sample chamber having an integralvent port within the chamber itself.

FIG. 28 is a cross-sectional view of one embodiment of a sample chamberfor extracting analyte from a solid or solid-containing matrix.

FIG. 29 is a cross-section view of an alternative embodiment of a samplechamber for extracting analyte from a solid or solid-containing matrixincorporating force focusing elements.

FIG. 30 is a cross-section view of another embodiment of a samplechamber for extracting analyte from a solid or solid-containing matrixincorporating a two-region, or compound, sample chamber.

FIG. 31 is a cross-sectional view depicting one embodiment of a bubbletrap chamber.

FIG. 32 is a schematic representation of another embodiment of an assaycartridge illustrating various fluidic components.

FIG. 33 is an exploded assembly drawing illustrating the laminarassemblage for a two-piece, extraction assay cartridge in accordancewith the schematic diagram given in FIG. 32.

FIG. 34 depicts a cutaway exploded view of one preferred design for acartridge reader.

DETAILED DESCRIPTION

The invention, as well as additional objects, features and advantagesthereof, will be understood more fully from the following detaileddescription of certain preferred embodiments. Where the terms “measure”or “measurement” are used herein, they are understood to encompassquantitative and qualitative measurement, and encompasses measurementscarried out for a variety of purposes including, but not limited to,detecting the presence of a thing or property, measuring the amount of athing or property, and/or identifying a thing or property in a sample

The present invention includes apparatuses, electrodes, electrodearrays, systems, system components, kits, reagents and methods forperforming one or more assays on a sample. The invention includes assaymodules (e.g., assay cartridges, assay plates, etc.) having one or moreassay cells (e.g., wells, compartments, chambers, conduits, flow cells,etc.) that may comprise one or more assay domains (e.g., discretelocations on a assay cell surface where an assay reaction occurs and/orwhere an assay dependent signal, such as an electrochemical orpreferably an electrode induced luminescence signal is induced) forcarrying out a plurality of assay measurements.

In certain preferred embodiments, assay domains are supported on assayelectrodes (preferably, an array of assay electrodes, most preferably aone dimensional array of assay electrodes) so as to permit the conductof assays based on electrochemical or electrode induced luminescencemeasurements. The assay domains are, optionally, defined by a dielectriclayer deposited on the electrodes. The assay modules, preferably, haveone or more attributes that make them suitable for use in “point ofcare” clinical measurements, e.g., small size, low cost, disposability,multiplexed detection, ease of use, etc. The methods and apparatuses ofthe invention, allow these benefits to be achieved while maintaining theperformance of traditional batch processing instruments of the typetypically used in the central clinical lab.

The assay module may comprise the necessary electronic components and/oractive mechanical components for carrying out an assay measurement,e.g., one or more sources of electrical energy, ammeters,potentiometers, light detectors, temperature monitors or controllers,pumps, valves, etc. Preferably, some or all of the electronic and/oractive mechanical components are arranged within a separate assay modulereader. The reader would also have the appropriate electrical, fluidicand/or optical connections to the assay module for carrying out an assayon the assay module. Using such an arrangement, the assay module can bedesigned to be low cost and disposable while the reader (which holds themore expensive and complex components) is reusable. A preferred assayprocedure using an assay module and assay reader would compriseinserting the cartridge in the reader, making the appropriateelectrical, fluidic and/or optical connections to the cartridge (makinguse of electrical, fluidic and/or optical connectors on the cartridgeand reader), and conducting an assay in the cartridge. The sample ispreferably introduced into the cartridge prior to inserting thecartridge in the reader. The assay may also involve adding one or moreassay reagents to the cartridge; preferably, one or more assay reagentsare stored in the cartridge in a dry and/or wet form.

The invention also includes methods of preparing the assay modulesincluding methods for preparing electrode arrays and forming assaydomains on these electrode arrays. The invention also includes methodsfor washing assay domains to remove unbound reagents without allowingthese reagents to interact with other surfaces in the assay module.

One preferred embodiment of the invention comprises an assay cartridgecomprising one or more assay flow cells. The assay flow cell comprises achamber having a fluid inlet and fluid outlet and a flow path betweenthe inlet and outlet. An array of electrodes is patterned on an internalsurface of the chamber. When used in electrode induced luminescenceassays, the internal chamber surface opposing the electrode array is,preferably, light-transmissive so as to allow for the detection of lightgenerated at the electrodes. One or more of the electrodes compriseassay reagents immobilized on the electrode. These assay domains areused to carry out assay reactions which are detected by using theelectrode to induce an assay dependent signal such as an electrochemicalor, more preferably, an electrode induced luminescence signal anddetecting the signal. Preferably, these assay reagents are arranged inone or more assay domains defined by apertures in a dielectric layerdeposited on the electrode. Optionally, the fluid inlet comprises afluid inlet line that has sensors for detecting the presence of fluid inthe fluid inlet line.

Preferably, the electrodes in the assay cartridge are patterned in a onedimensional array along the fluid path. The array and or fluid path are,preferably, in a linear arrangement, although other shapes (e.g., arcs,curves, zig-zags, etc. may also be used). In such a configuration, it isadvantageous for the active area of the electrodes and aspect ratio ofthe flow path be selected to ensure that assay domains on the electrodeefficiently sample analytes in fluids passing through the flow cell.Most preferably, the length of the flow path along the direction of flowis greater than the width perpendicular to the direction of flow, theactive area of the electrode takes up a significant portion of the widthof the flow path (preferably greater than 60%, more preferably greaterthan 80%), and/or the height of the flow path above the electrodes issmall compared to the width of the flow path. Surprisingly, it has beenfound that the surface area of dedicated counter electrodes in the flowcell can be reduced significantly without affecting assay performance byreusing electrodes used as working electrodes (e.g., working electrodeshaving binding domains used for electrode induced luminescence assays),these electrodes being reused as counter electrodes for measuring anassay dependent signal from another, preferably adjacent, workingelectrode. In an especially preferred embodiment, the electrodes areactivated in a pair-wise fashion along the path of the flow cell, theinterior electrodes in the one-dimensional electrode array being used asworking electrodes for inducing an assay dependent signal andsubsequently as counter electrodes for inducing an assay dependentsignal at an adjacent electrode.

The assay cartridges of the invention may comprise a plurality of flowcells or detection chambers. In certain preferred embodiments the flowcell may comprise the same assay domains or, at least, have at leastsome assay domains that share specificity for the same analytes ofinterest. In these embodiments, the plurality of flow cells may be usedto analyze a plurality of different samples or to compare samples thathave been pre-treated in different ways. Alternatively, one of the flowcells may be a control flow cell used to analyze a control sample andanother of the flow cells may be a test flow cell used to analyze a testsample. The control sample may be a completely pre-defined controlsample or may be a mixture comprising the test sample but spiked withadded analytes of interest so as to allow for calibration of the assaysby the method of standard addition. In an alternative embodiment, theassay cartridge has at least two flow cells that have assay domains fortwo different assay panels. Advantageously, such a cartridge may be usedto separately perform assay reactions that are incompatible with eachother.

FIG. 1 a depicts a simplified schematic of a cartridge-based biochemicaldetection system 100 in accordance with one embodiment of the invention.Preferably a system housing, e.g., cartridge reader 105, would includean optical detector 110 and would be adapted and configured to receiveand position cartridge 115 and/or optical detector 110 for processing.The system would preferably contain support subsystems (not shown) thatmay include one or more of the following: storage subsystem for storingassay reagents/consumables and/or waste; sampleacquisition/preprocessing/storage subsystem for sample handling; fluidichandling subsystem for handling the reagents, sample, waste, etc. andfor providing fluids to the detection chamber 120 via a fluid inlet line125; electrical subsystem for electrically contacting the cartridge'selectrical contacts 130 and supplying electrical energy to theelectrodes 135,136,137; and a control subsystem for controlling andcoordinating operation of the system and subsystems and for acquiring,processing and storing the optical detection signal.

As illustrated, one preferred embodiment would use an electrode arraythat preferably has at least one dedicated counter electrode 135, onedual-role electrode 136 and one dedicated working electrode 137. Such apreferred configuration would use a pair-wise firing scheme (discussedin detail below) wherein the dual-role electrode can be reused. FIG. 1 bdepicts in greater detail one possible embodiment for the detectionportion of a cartridge-based device 150. As depicted, two detectionchambers 155,156 each contain a bank of nine individually addressableelectrodes 157,158. There are two fluid input lines depicted 160,161 forintroducing sample, reagents and/or wash solutions into the detectionchambers and two banks of electrical contacts 165,166 with correspondingelectrical leads 170,171 to the electrodes 157,158. Also depicted inthis preferred embodiment are two banks of impedance sensors 172,173that may be used fluid detection (e.g., sample, reagents, wash, buffer,etc.) and/or fluid discrimination (e.g., discriminating between sample,reagents, wash, buffer, etc. and/or sample type such as whole blood,plasma, mucous, etc.).

FIG. 1 c is an assembly schematic for one preferred embodimentillustrating the assembly of cartridge component 178 comprising anelectrode array 176. According to one embodiment, electrode array 176(preferably, comprised of carbon ink) is applied to the substrate layer175 forming the electrode 180, electrical lead 181 and electricalcontact 182 portions. A dielectric layer 177 is preferably applied overthe electrode layer to define the assay domains 190 and the impedancesensors 191. Alternately, electrical contacts 182 could be printed onthe opposing side of the substrate and connected to electrodes 180 orelectrical leads 181 via conductive through-holes through the substrate.Methods for applying the carbon and dielectric layers as well as variousalternative materials are discussed below in greater detail.

Cartridge component 178 is, preferably, mated with a second cartridgecomponent. The second cartridge component has channels or aperturesarranged on the mating surface so that when mated to cartridge component178 it acts to form detection chambers over the electrode arrays (e.g.,as illustrated by detection chambers 155 and 156 in FIG. 1 b anddetection chamber 120 in FIG. 1 a). Preferably, the second cartridgecomponent has channels on the mating surface that form flow cells overthe electrodes when mated to component 178 (the flow cells having onesurface defined by component 178 and an opposing surface and wellsdefined by the second component. The channels may also be used to formother fluidic paths such as fluidic inlet and outlet lines to the flowcell. These channels may, e.g., be molded or cut into the secondcomponent. Alternatively, the walls of the flow cell or other fluidicpaths may be defined by a gasket material preferably, double sidedadhesive tape) applied between component 178 and the second cartridgecomponent. Alternatively, the second component has apertures in themating surface that form wells when mated to component 178.

In a preferred embodiment of the invention, an assay cartridge hasminimal or no active mechanical or electronic components. When carryingout an assay, such an assay cartridge may be introduced into a cartridgereader which provides these functions. For example, a reader may haveelectronic circuitry for applying electrical energy to the assayelectrodes and for measuring the resulting potentials or currents atassay electrodes. The reader may have one or more light detectors formeasuring luminescence generated at assay electrodes. Light detectorsthat may be used include, but are not limited to photomultiplier tubes,avalanche photodiodes, photodiodes, photodiode arrays, CCD chips, CMOSchips, film. The light detector may be comprised within an opticaldetection system that also comprise lenses, filters, shutters,apertures, fiber optics, light guides, etc. The reader may also havepumps, valves, heaters, sensors, etc. for providing fluids to thecartridge, verifying the presence of fluids and/or maintaining thefluids at an appropriate controlled temperature. The reader may be usedto store and provide assay reagents, either onboard the reader itself orfrom separate assay reagent bottles or an assay reagent storage device.The reader may also have cartridge handling systems such as motioncontrollers for moving the cartridge in and out of the reader. Thereader may have a microprocessor for controlling the mechanical and/orelectronic subsystems, analyzing the acquired data and/or providing agraphical user interface (GUI). The cartridge reader may also compriseelectrical, mechanical and/or optical connectors for connecting to thecartridge.

One aspect of the invention relates to the assay modules employingelectrodes, the immobilization of assay reagents on these electrodes,and their use in assays, preferably electrode-induced luminescenceassays. Co-pending U.S. patent application Ser. No. 10/185,274, filedJun. 28, 2002, hereby incorporated by reference, provides a number ofexamples of electrode and dielectric materials, electrode patterns andpatterning techniques and immobilization techniques that are adapted foruse in electrode-induced luminescence assays and suitable for use withthe assay modules of the invention. Electrodes in the present inventionare preferably comprised of a conductive material. The electrode maycomprise a metal such as gold, silver, platinum, nickel, steel, iridium,copper, aluminum, a conductive alloy, or the like. They may alsocomprise oxide coated metals (e.g. aluminum oxide coated aluminum).Electrodes may comprise non-metallic conductors such as conductive formsof molecular carbon. Electrodes may also be comprised of semiconductingmaterials (e.g. silicon, germanium) or semi-conducting films such asindium tin oxide (ITO), antimony tin oxide (ATO) and the like.Electrodes may also be comprised of mixtures of materials containingconductive composites, inks, pastes, polymer blends, metal/non-metalcomposites and the like. Such mixtures may include conductive orsemi-conductive materials mixed with non-conductive materials.Preferably, electrode materials are substantially free of silicone-basedmaterials.

Electrodes (in particular working electrodes) used in assay modules ofthe invention are advantageously able to induce luminescence fromluminescent species. Preferable materials for working electrodes arematerials able to induce electrochemiluminescence fromruthenium-tris-bipyridine in the presence of tertiary alkyl amines (suchas tripropyl amine). Examples of such preferred materials includeplatinum, gold, ITO, carbon, carbon-polymer composites, and conductivepolymers.

Preferably, electrodes are comprised of carbon-based materials such ascarbon, carbon black, graphitic carbon, carbon nanotubes, carbonfibrils, graphite, carbon fibers and mixtures thereof. Advantageously,they may be comprised of conductive carbon-polymer composites,conductive particles dispersed in a matrix (e.g. carbon inks, carbonpastes, metal inks), and/or conductive polymers. One preferredembodiment of the invention is an assay module, preferably an assaycartridge, having electrodes (e.g., working and/or counter electrodes)that comprise carbon, preferably carbon layers, more preferablyscreen-printed layers of carbon inks. Some useful carbon inks includematerials produced by Acheson Colloids Co. (e.g., Acheson 440B, 423ss,PF407A, PF407C, PM-003A, 30D071, 435A, Electrodag 505SS, and Aquadag™),E. I. Du Pont de Nemours and Co. (e.g., Dupont 7105, 7101, 7102, 7103,7144, 7082, 7861D, E100735 62B and CB050), Advanced Conductive Materials(e.g., PTF 20), Gwen Electronics Materials (e.g., C2000802D2) andConductive Compounds Inc (e.g., C-100), and Ercon Inc. (e.g., G-451,G-449 and 150401).

In another preferred embodiment, the electrodes of the inventioncomprise carbon fibrils. The terms “carbon fibrils”, “carbon nanotubes”,single wall nanotubes (SWNT), multiwall nanotubes (MWNT), “graphiticnanotubes”, “graphitic fibrils”, “carbon tubules”, “fibrils” and“buckeytubes”, all of which terms may be used to describe a broad classof carbon materials (see Dresselhaus, M. S.; Dresselhaus, G.; Eklund, P.C.; “Science of Fullerenes and Carbon Nanotubes”, Academic Press, SanDiego, Calif., 1996, and references cited therein). The terms “fibrils”and “carbon fibrils” are used throughout this application to includethis broad class of carbon-based materials. Individual carbon fibrils asdisclosed in U.S. Pat. Nos. 4,663,230; 5,165,909; and 5,171,560 areparticularly advantageous. They may have diameters that range from about3.5 nm to 70 nm, and length greater than 10² times the diameter, anouter region of multiple, essentially continuous, layers of orderedcarbon atoms and a distinct inner core region. Simply for illustrativepurposes, a typical diameter for a carbon fibril may be approximatelybetween about 7 and 25 nm, and a typical range of lengths may be 1000 nmto 10,000 nm. Carbon fibrils may also have a single layer of carbonatoms and diameters in the range of 1 nm-2 nm. Electrodes of theinvention may comprise one or more carbon fibrils, e.g., in the form ofa fibril mat, a fibril aggregate, a fibril ink, a fibril composite(e.g., a conductive composite comprising fibrils dispersed in an oil,paste, ceramic, polymer, etc.).

Electrodes may be formed into patterns by a molding process (i.e.,during fabrication of the electrodes), by patterned deposition, bypatterned printing, by selective etching, through a cutting process suchas die cutting or laser drilling, and/or by techniques known in the artof electronics microfabrication. Electrodes may be self supporting ormay be supported on another material, e.g. on films, plastic sheets,adhesive films, paper, backings, meshes, felts, fibrous materials, gels,solids (e.g. metals, ceramics, glasses), elastomers, liquids, tapes,adhesives, other electrodes, dielectric materials and the like. Thesupport, or substrate, may be rigid or flexible, flat or deformed,transparent, translucent, opaque or reflective. Preferably, the supportcomprises a flat sheet of plastic such as acetate or polystyrene.Electrode materials may be applied to a support by a variety of coatingand deposition processes known in the art such as painting,spray-coating, screen-printing, ink-jet printing, laser printing,spin-coating, evaporative coating, chemical vapor deposition, etc.Supported electrodes may be patterned using photolithographic techniques(e.g., established techniques in the microfabrication of electronics),by selective etching, and/or by selective deposition (e.g., byevaporative or CVD processes carried out through a mask). In a preferredembodiment, electrodes are comprised of extruded films of conductingcarbon/polymer composites. In another preferred embodiment, electrodesare comprised of a screen printed conducting ink deposited on asubstrate. Electrodes may be supported by another conducting material.In some applications, screen printed carbon ink electrodes are printedover a conducting metal ink (e.g., silver ink) layer so as to improvethe conductivity of the electrodes. Preferably, in assay cartridges, aminiaturized design allows the use of electrodes having short printedelectrode leads (preferably less than 1.5 cm, more preferably less than1.0 cm) that are relatively similar in length. By keeping the leadsshort, it is possible to use screen printed carbon electrodes without anunderlying conductive metal layer such as a silver layer.

According to one preferred embodiment of the invention, the electrodesurface (preferably a working electrode surface of an assay module orassay plate) is bounded by a dielectric surface, the dielectric surfacebeing raised or lowered preferably, raised) and/or of differenthydrophobicity (preferably, more hydrophobic) than the electrodesurface. Preferably, the dielectric boundary is higher, relative to theelectrode surface, by 0.5-100 micrometers, or more preferably by 2-30micrometers, or most preferably by 8-12 micrometers. Even morepreferably, the dielectric boundary has a sharply defined edge (i.e.,providing a steep boundary wall and/or a sharp angle at the interfacebetween the electrode and the dielectric boundary).

Preferably, the first electrode surface has an advancing contact anglefor water 10 degrees less than the dielectric surface, preferably 15degrees less, more preferably 20 degrees less, more preferably 30degrees less, even more preferably 40 degrees less, and most preferred50 degrees less. One advantage of having a dielectric surface that israised and/or more hydrophobic than the electrode surface is in thereagent deposition process where the dielectric boundary may be used toconfine a reagent within the boundary of the electrode surface. Inparticular, having a sharply defined edge with a steep boundary walland/or a sharp angle at the interface between the electrode anddielectric boundary is especially useful for “pinning” drops of solutionand confining them to the electrode surface. In an especially preferredembodiment of the invention, the dielectric boundary is formed byprinting a patterned dielectric ink on and/or around the electrode, thepattern designed so as to expose one or more assay domains on theelectrode.

Electrodes may be modified by chemical or mechanical treatment toimprove the immobilization of reagents. The surface may be treated tointroduce functional groups for immobilization of reagents or to enhanceits adsorptive properties. Surface treatment may also be used toinfluence properties of the electrode surface, e.g., the spreading ofwater on the surface or the kinetics of electrochemical processes at thesurface of the electrode. Techniques that may be used include exposureto electromagnetic radiation, ionizing radiation, plasmas or chemicalreagents such as oxidizing agents, electrophiles, nucleophiles, reducingagents, strong acids, strong bases and/or combinations thereof.Treatments that etch one or more components of the electrodes may beparticularly beneficial by increasing the roughness and therefore thesurface area of the electrodes. In the case of composite electrodeshaving conductive particles or fibers (e.g., carbon particles orfibrils) in a polymeric matrix or binder, selective etching of thepolymer may be used to expose the conductive particles or fibers.

One particularly useful embodiment is the modification of the electrode,and more broadly a material incorporated into the present invention bytreatment with a plasma, specifically a low temperature plasma, alsotermed glow-discharge. The treatment is carried out in order to alterthe surface characteristics of the electrode, which come in contact withthe plasma during treatment. Plasma treatment may change, for example,the physical properties, chemical composition, or surface-chemicalproperties of the electrode. These changes may, for example, aid in theimmobilization of reagents, reduce contaminants, improve adhesion toother materials, alter the wettability of the surface, facilitatedeposition of materials, create patterns, and/or improve uniformity.Examples of useful plasmas include oxygen, nitrogen, argon, ammonia,hydrogen, fluorocarbons, water and combinations thereof. Oxygen plasmasare especially preferred for exposing carbon particles in carbon-polymercomposite materials. Oxygen plasmas may also be used to introducecarboxylic acids or other oxidized carbon functionality into carbon ororganic materials (these may be activated, e.g., as active esters oracyl chlorides) so as to allow for the coupling of reagents. Similarly,ammonia-containing plasmas may be used to introduce amino groups for usein coupling to assay reagents.

Treatment of electrode surfaces may be advantageous so as to improve orfacilitate immobilization, change the wetting properties of theelectrode, increase surface area, increase the binding capacity for theimmobilization of reagents (e.g., lipid, protein or lipid/proteinlayers) or the binding of analytes, and/or alter the kinetics ofelectrochemical reactions at the electrode. In some applications,however, it may be preferable to use untreated electrodes. For example,we have found that it is advantageous to etch carbon ink electrodesprior to immobilization when the application calls for a large dynamicrange and therefore a high binding capacity per area of electrode. Wehave discovered that oxidative etching (e.g., by oxygen plasma) hasadditional advantages in that the potential for oxidation of tripropylamine (TPA) and the contact angle for water are both reduced relative tothe unetched ink. The low contact angle for water allows reagents to beadsorbed on the electrode by application of the reagents in a smallvolume of aqueous buffer and allowing the small volume to spread evenlyover the electrode surface. Surprisingly, we have found that excellentassays may also be carried out on unetched carbon ink electrodes despitethe presence of polymeric binders in the ink. In fact, in someapplications requiring high sensitivity or low-non specific binding itis preferred to use unetched carbon ink electrodes so as to minimize thesurface area of exposed carbon and therefore minimize background signalsand loss of reagents from non-specific binding of reagents to theexposed carbon. Depending on the ink used and the process used to applythe ink, the electrode surface may not be easily wettable by aqueoussolutions. We have found that we can compensate for the low wettabilityof the electrodes during the adsorption of reagents by adding lowconcentrations of non-ionic detergents to the reagent solutions so as tofacilitate the spreading of the solutions over the electrode surface.Even spreading is especially important during the localizedimmobilization of a reagent from a small volume of solution. Forexample, we have found that the addition of 0.005-0.04% Triton X-100®allows for the spreading of protein solutions over unetched carbon inksurfaces without affecting the adsorption of the protein to theelectrode and without disrupting the ability of a dielectric filmapplied on or adjacent to the electrode (preferably, a printeddielectric film with a thickness of 0.5-100 micrometers, or morepreferably 2-30 micrometers, or most preferably 8-12 micrometers andhaving a sharply defined edge) to confine fluids to the electrodesurface. Preferably, when non-ionic detergents such as Triton X-100® areused to facilitate spreading of reagents (e.g., capture reagents) ontounetched screen-printed electrodes (i.e., so as to allow theimmobilization of the reagents), the solutions containing the reagentsare allowed to dry onto the electrode surface. It has been found thatthis drying step greatly improves the efficiency and reproducibility ofthe immobilization process.

The efficiency of the immobilization of reagents on carbon inkelectrodes, especially unetched carbon ink electrodes, may exhibit somevariability due to different levels of contamination of the electrodessurface. This effect is particularly pronounced when certain dielectricinks are used to form assay domains on the electrodes. We have foundthat we can improve the immobilization efficiencies and lower thevariability by pre-washing the electrode surfaces, preferably with asurfactant solution.

The contamination of carbon ink electrodes by certain dielectric inkswas observed by quantitatively assessing the surface wetting propertiesof the electrodes by measuring the contact diameter, where the largerthe contact diameter, the better the wetting. A comparison of threealternative carbon surfaces with different dielectric layers is depictedin Table 1. As shown by the data in Table 1, washing the electrodesurfaces can significantly increase the wetting properties (contactdiameter) of carbon surfaces contacting the 451 dielectric (presumablyby removing contamination of the electrode surface associated with theprinting of the 451 dielectric, e.g., by migration of components of thedielectric ink on to the electrode surface).

TABLE 1 Comparison of Contact Diameters on Carbon Electrode Surfaces forThree Different Dielectric Materials (Mean 50 nL water drop diameter at400 μs open time) Surface Contact Diameter, inches * No pre-treatment:Carbon with 451 dielectric 0.0366 Carbon with Nazdar dielectric 0.0461Carbon with PD039A dielectric 0.0457 Pre-treated: Carbon with 451dielectric 0.0438 Carbon with Nazdar dielectric 0.0463 Carbon withPD039A dielectric 0.0448

In one embodiment, a method of decontaminating the carbon electrodesurfaces may be employed wherein the electrode surfaces are soaked in anaqueous 0.5% Triton X-100 solution for several hours, subsequentlyrinsed with deionized water, then soaked in deionized water forapproximately one hour and finally dried. The Triton solution preferablyremoves the contaminants from the surface and the deionized waterremoves the adsorbed surfactant. This method of decontamination is aneffective cleaning procedure that enhances the differences between theretreating contact angles on the carbon and the dielectric inks.

FIG. 6 demonstrates the results of the decontamination procedure.Specifically, FIG. 6 depicts images of ECL from an ECL label over carbonink electrodes, the exposed areas of the electrode being defined by adielectric film. FIG. 6 a is the ECL image without decontamination andFIG. 6 b is the ECL image after decontamination with Triton X-100 inaccordance with the present embodiment. These ECL images show that thetreatment process greatly reduces the variation in ECL intensity overthe surface of the electrode, the patchiness of ECL on the untreatedelectrode presumably being caused by patches of contamination on thesurface.

Electrodes can be derivatized with chemical functional groups that canbe used to attach other materials to them. Materials may be attachedcovalently to these functional groups, or they may be adsorbednon-covalently to derivatized or underivatized electrodes. Electrodesmay be prepared with chemical functional groups attached covalently totheir surface. These chemical functional groups include but are notlimited to COOH, OH, NH₂, activated carboxyls (e.g., N-hydroxysuccinimide (NHS)-esters), poly-(ethylene glycols), thiols, alkyl((CH₂)_(n)) groups, and/or combinations thereof). Certain chemicalfunctional groups (e.g., COOH, OH, NH₂, SH, activated carboxyls) may beused to couple reagents to electrodes. For further reference to usefulimmobilization and bioconjugation techniques see G. Hermanson, A. Malliaand P. Smith, Immobilized Affinity Ligand Techniques (Academic Press,San Diego, 1992) and G. Hermanson, Bioconjugate Techniques (AcademicPress, San Diego, 1996).

In preferred embodiments, NHS-ester groups are used to attach othermolecules or materials bearing a nucleophilic chemical functional group(e.g., an amine). In a preferred embodiment, the nucleophilic chemicalfunctional group is present on and/or in a biomolecule, either naturallyand/or by chemical derivatization. Examples of suitable biomoleculesinclude, but are not limited to, amino acids, proteins and functionalfragments thereof, antibodies, binding fragments of antibodies, enzymes,nucleic acids, and combinations thereof. This is one of many suchpossible techniques and is generally applicable to the examples givenhere and many other analogous materials and/or biomolecules. In apreferred embodiment, reagents that may be used for ECL may be attachedto the electrode via NHS-ester groups.

It may be desirable to control the extent of non-specific binding ofmaterials to electrodes. Simply by way of non-limiting examples, it maybe desirable to reduce or prevent the non-specific adsorption ofproteins, antibodies, fragments of antibodies, cells, subcellularparticles, viruses, serum and/or one or more of its components, ECLlabels (e.g., Ru^(II)(bpy)₃ and R^(III)(bpy)₃ derivatives), oxalates,trialkylamines, antigens, analytes, and/or combinations thereof). Inanother example, it may be desirable to enhance the binding ofbiomolecules.

One or more chemical moieties that reduce or prevent non-specificbinding (also known as blocking groups) may be present in, on, or inproximity to an electrode. Such moieties, e.g., PEG moieties and/orcharged residues (e.g., phosphates, ammonium ions), may be attached toor coated on the electrode. Examples of useful blocking reagents includeproteins (e.g., serum albumins and immunoglobins), nucleic acids,polyethylene oxides, polypropylene oxides, block copolymers ofpolyethylene oxide and polypropylene oxide, polyethylene imines anddetergents or surfactants (e.g., classes of non-ionicdetergents/surfactants known by the trade names of Brij, Triton, Tween,Thesit, Lubrol, Genapol, Pluronic (e.g., F108), Tetronic, Tergitol, andSpan).

Materials used in electrodes may be treated with surfactants to reducenon-specific binding. For example, electrodes may be treated withsurfactants and/or detergents that are well known to one of ordinaryskill in the art (for example, the Tween, Triton, Pluronics (e.g.,F108), Span, and Brij series of detergents). Solutions of PEGs and/ormolecules which behave in similar fashion to PEG (e.g., oligo- orpolysaccharides, other hydrophilic oligomers or polymers) (“Polyethyleneglycol chemistry: Biotechnical and Biomedical Applications”, Harris, J.M. Editor, 1992, Plenum Press) may be used instead of and/or inconjunction with surfactants and/or detergents. Undesirable non-specificadsorption of certain entities such as those listed above may be blockedby competitive non-specific adsorption of a blocking agent, e.g., by aprotein such as bovine serum albumin (BSA), casein or immunoglobulin G(IgG). One may adsorb or covalently attach an assay reagent on anelectrode and subsequently treat the electrode with a blocking agent soas to block remaining unoccupied sites on the surface.

In preferred embodiments, it may be desirable to immobilize (by eithercovalent or non-covalent means) biomolecules or other assay reagents tocarbon-containing materials, e.g., carbon inks, carbon black, fibrils,and/or carbon dispersed in another material. One may attach antibodies,fragments of antibodies, proteins, enzymes, enzyme substrates,inhibitors, cofactors, antigens, haptens, lipoproteins, liposaccharides,cells, sub-cellular components, cell receptors, viruses, nucleic acids,antigens, lipids, glycoproteins, carbohydrates, peptides, amino acids,hormones, protein-binding ligands, pharmacological agents, and/orcombinations thereof. It may also be desirable to attach non-biologicalentities such as, but not limited to polymers, elastomers, gels,coatings, ECL tags, redox active species (e.g., tripropylamine,oxalates), inorganic materials, chelating agents, linkers, etc. Aplurality of species may be co-adsorbed to form a mixed layer on thesurface of an electrode. Most preferably, biological materials (e.g.,proteins) are immobilized on carbon-containing electrodes by passiveadsorption. Surprisingly, biological membranes (e.g., cells, cellmembranes, membrane fragments, membrane vesicles, lipsomes, organelles,viruses, bacteria, etc.) may be directly adsorbed on carbon withoutdestroying the activity of membrane components or their accessibility tobinding reagents (see, e.g., copending U.S. patent application Ser. No.10/208,526 (entitled “Assay Electrodes Having Immobilized Lipid/ProteinLayers, Methods Of Making The Same And Methods Of Using The Same ForLuminescence Test Measurements”), filed on Jul. 29, 2002, herebyincorporated by reference.

Electrodes used in the assay modules are, preferably, non-porous,however, in some applications it is advantageous to use porouselectrodes (e.g., mats of carbon fibers or fibrils, sintered metals, andmetals films deposited on filtration membranes, papers or other poroussubstrates. These applications include those that employ filtration ofsolutions through the electrode so as to: i) increase mass transport tothe electrode surface (e.g., to increase the kinetics of binding ofmolecules in solution to molecules on the electrode surface); ii)capture particles on the electrode surface; and/or iii) remove liquidfrom the well.

Preferred assay modules may use dielectric inks, films or otherelectrically insulating materials (hereinafter referred to asdielectrics). Dielectrics in the present invention may be used toprevent electrical connectivity between electrodes, to define patternedregions, to adhere materials together (i.e., as adhesives), to supportmaterials, to define assay domains, as masks, as indicia and/or tocontain assay reagents and other fluids. Dielectrics are non-conductingand advantageously non-porous (i.e., do not permit transmission ofmaterials) and resistant to dissolving or degrading in the presence ofmedia encountered in an electrode induced luminescence measurement. Thedielectrics in the present invention may be liquids, gels, solids ormaterials dispersed in a matrix. They may be deposited in uncured formand cured to become solid. They may be inks, solid films, tapes orsheets. Materials used for dielectrics include polymers, photoresists,plastics, adhesives, gels, glasses, non-conducting inks, non-conductingpastes, ceramics, papers, elastomers, silicones, thermoplastics.Preferably, dielectric materials of the invention are substantially freeof silicones. Examples of non-conducting inks include UV curabledielectrics such as materials produced by Acheson Colloids Co. (e.g.,Acheson 451SS, 452SS, PF-455, PD039A, PF-021, ML25251, ML25240, ML25265,and Electrodag 38DJB136 clear), Nazdar (e.g., Nazdar GS2081 3400SPL) andE. I. du Pont de Nemours and Co. (e.g., Dupont: 5018, 3571, and 5017).

Dielectrics, in accordance with certain preferred embodiments, may beapplied by a variety of means, for example, printing, spraying,laminating, or may be affixed with adhesives, glues, solvents or by useof mechanical fasteners. Patterns and/or holes in dielectric layers maybe formed by molding processes (i.e., during fabrication of the layer),by selective etching and/or by a cutting process such as die cutting orlaser drilling. Dielectrics may be deposited and/or etched in patternsthrough the use of established photolithographic techniques (e.g.,techniques used in the semiconductor electronics industry) and/or bypatterned deposition using an evaporative or CVD process (e.g., bydeposition through a mask). In a preferred embodiment, a dielectric inkis deposited on a substrate by printing (e.g., ink jet printing, laserprinting or, more preferably, screen printing) and, optionally, UVcured. Preferably, the screen printed dielectric is UV curable allowingfor improved edge definition than solvent based dielectrics. In anotherpreferred embodiment, a non-conducting polymeric film is affixed to asupport using an adhesive.

When using a dielectric ink printed on, or adjacent to, an electrode toconfine fluids to regions of the electrode surface, the dielectric filmpreferably has a thickness of 0.5-100 micrometers, or more preferably2-30 micrometers, or most preferably 8-12 micrometers and also,preferably, has a sharply defined edge with steep walls.

Miniaturization of various components and processes required to supportECL-based assays can also benefit from novel approaches to induce ECL.When inducing ECL, the working electrode and a counter electrode are,preferably, spaced relatively close to one another to minimize theeffect of voltage drops in solution on the intensity and spatialdistribution of ECL signals. When multiple ECL measurements are to bemade in the same solution volume, each measurement, preferably, uses aclosely spaced working electrode (where electrochemiluminescence isinduced) and a counter electrode (to complete the electrochemicalcircuit). One possible configuration is for each measurement to have itsown pair of electrodes; however, this configuration would require thelargest volume, space, and number of electrical contacts on the device.An alternative configuration is for each measurement to share a commoncounter electrode that is reused. FIGS. 3 f and 3 g illustrate possiblealternative approaches for using common counter electrodes. As can beseen, the detection chambers (e.g., detection chamber 341) for suchconfigurations would still require a large space in order to accommodateboth the working electrodes (e.g., working electrode 315) and thesingle, common counter electrode 311. Moreover, the relative size andspacing of each working electrode-counter electrode pair will affect therelative performance of each pair. Therefore, as depicted in FIGS. 3 fand 3 g configurations employing a single, common counter electrodewould preferably ensure that the relative size and spacing of eachworking-counter electrode pair is approximately equal. Preferably, theworking electrodes are arranged in a one dimensional array, the arraybeing preferably arranged along the flow path of a flow cell. The commoncounter electrode is also, preferably aligned with the flow path to oneside of the array so as to maintain approximate equal spacing to each ofthe working electrodes. Preferably, no working electrode is located inthe shortest path between the counter electrode and a different workingelectrode; application of a large potential between the counterelectrode and a first working electrode can under some conditionsgenerate high enough potentials in the intervening solution to triggeran undesired emission of ECL at a second working electrode located inthe shortest path between the first working electrode and the counterelectrode. Optionally, the electrode surface area in contact with thedetection chamber is defined by an aperture in a dielectric filmdeposited on the electrode layer (shown as circles on the electrodelayer).

In one preferred embodiment, an electrode pair-wise firing scheme can beemployed in order to miniaturize the cartridge to the largest extentpracticable, and therefore greatly reduce the volume and space required.This preferred pair-wise firing scheme, or electrode-pairing scheme,would preferably employ a sacrificial, or dedicated counter electrodefor the first measurement and thereafter allow the reuse of a previouslyfired (where fired describes the state of the surface after theapplication of a working electrode potential, e.g., a potentialsufficient to generate electrochemiluminescence at a working electrode)working electrode as the next counter electrode for the nextmeasurement. Surprisingly, as discussed below, it was observed thatneither having a protein coating on the electrode being used as thecounter electrode nor the fact that the electrode was already fired onceas a working electrode affected the performance of that electrode foruse as a counter electrode, thus allowing the use of electrodes in adual-role as both working and counter electrodes.

FIGS. 3 a-3 e depict possible alternative configurations for electrodearrays employing the pair-wise firing scheme. FIG. 3 a illustrates asingle bank of electrodes that can be used in one or more detectionchambers (a single detection chamber 340 is indicated here by the dottedline). The electrodes are preferably arranged in a one dimensionalarray. Optionally, the electrode surface area in contact with thedetection chamber is defined by an aperture in a dielectric filmdeposited on the electrode layer (shown as circles on the electrodelayer). In one embodiment, electrode 310 may be configured as thededicated counter electrode, electrodes 305-309 may be configured as thedual-role electrodes and electrode 315 may be configured as thededicated working electrode. The electrode bank has impedance sensors325 on leads to the electrodes which can be arranged to contact fluid ininput or outlet lines to the detection chamber. Preferably, theimpedance sensors are defined by apertures in a dielectric layerdeposited on the electrode layer. The electrode array of FIG. 3 autilizes a configuration wherein the electrical contacts and leads arelocated to one side of the electrodes allowing for simplified matingwith the control unit. FIG. 3 b depicts an alternative configurationwherein the electrical contacts and leads are alternately placed oneither side of the electrodes. Such an alternating configuration canallow for the impedance sensors to be placed on each of the electricalleads so as to allow interrogation of the fluids during both ingress andegress from the detection chamber (e.g., by arranging the fluid inletline and fluid outlet line so that they, respectively, contact impedancesensors on alternate sides of the electrodes).

FIGS. 3 c-3 e illustrate configurations employing multiple detectionchambers. In particular, FIGS. 3 c and 3 d depict two detection chambersemploying two banks of electrodes. FIG. 3 d illustrates a configurationwherein the electrodes for one set of contacts/leads are within theoppositely placed detection chamber. Such a configuration may provideadded benefits such as a more densely packed electrode array and theability to place impedance sensors on each lead. Impedance sensors maybe placed on each lead since each detection chamber can be alternatelyprocessed; i.e., fluid is first directed to on detection chamber and allassays are performed and then fluid is directed to the other detectionchamber for processing of the remaining assays.

FIG. 3 e depicts an embodiment utilizing four detection chambers. Itshould be noted that while FIG. 3 e depicts an electrode array employinga single, common counter electrode in each detection chamber, such aconfiguration can also be employed using the pair-wise firing schemediscussed above.

Preferably, the electrode arrays depicted in FIGS. 3 a-3 g are supportedon a support such as a plastic film or sheet. The detection chambersare, preferably, formed by mating the support to a second cartridgecomponent having channels or apertures defined thereon (optionally,these features being at least partially defined by a gasket between theelectrode support and the second cartridge component); see thediscussion of FIG. 1 c.

Since it was believed that using the electrode-pairing scheme mightresult in the assay on a previously used working electrode affecting itsfunction as the counter electrode for the next working electrode, anexperiment was devised wherein three different protein coatings wereused to determine their effect. The effects of three protein coatingswere measured: avidin, CK-MB capture antibody, and Bovine IgG. The ECLof a 10 nM ruthenium-tris-bipyridine solution in a tripropylamine-containing buffer was measured on non-coated electrodes withvarious counter electrodes (coated, non-coated, fired, and virgin);these results are listed in Table 2. In this table ECL_(fired CE)denotes the ECL from the working electrode when paired with a counterelectrode that has been previously fired as a working electrode andECL_(virgin CE) is for ECL from the working electrode when paired with acounter electrode that has not been previously fired as a workingelectrode. The observed ECL signals were all within experimental errorof one another demonstrating the unexpected result that neither thepresence of protein on the surface nor the prior use as a workingelectrode had any affect on the performance of that surface as a counterelectrode.

TABLE 2 Effects of Protein Coating and Application of OxidativePotentials to Electrodes Previously Used as a Counter Electrode in FreeTAG ECL Generation Protein on C.E. ECL_(fired CE) ECL_(virgin CE)anti-CK-MB 199 207 Blank 199 197 Avidin 181 205 IgG 203 214

With reference to FIG. 4, and by way of example only, operation of asimplified electrode array employing the pair-wise firing scheme withina single detection chamber will be described. For purposes of thisoperational example, introduction of sample, assay reagent(s), washsolution(s) and/or buffer(s) through the fluid input line 450 will notbe discussed; it is to be understood that each of the necessaryconstituents for performing the assay are present in the detectionchamber for this example. At least one of the electrodes will operate asa dedicated counter electrode, e.g., 401, and will therefore not haveany assay reagents immobilized thereon. Electrodes 402-407 will haveassay reagents immobilized thereon; electrodes 402-406 are to be used asdual-role electrodes and electrode 407 is to be used as a dedicatedworking electrode. As pictured in the figure, the electrodes arepreferably arranged in one dimensional arrays (most preferably, lineararrays) along the fluid path in the detection chamber. The dedicatedcounter electrode 401 will be used first in conjunction with theadjacent dual-role electrode 402, wherein the dual-role electrode willbe operated as a working electrode to perform the desired assay atdual-role electrode 402. Thereafter, dual-role electrode 402 will beoperated as a counter electrode and will be pair-wise fired withdual-role electrode 403, wherein dual-role electrode 403 will beoperated as a working electrode to perform the desired assay atdual-role electrode 403. This pair-wise firing is continued for theremaining electrodes until electrode pair 406 and 407. This lastremaining pair will operate dual-role electrode 406 as a counterelectrode and dedicated working electrode 407 as a working electrode toperform the desired assay at dedicated working electrode 407.Preferably, the electrode pairs used in a specific firing are adjacenteach other (i.e., there are no other electrodes located between them) toavoid the undesired emission of ECL from an electrode located in theintervening space.

The use of patterned electrodes in cartridges may impose certain uniquedesign and/or performance constraints. In particular, the use ofpatterned electrode leads may lead to problems associated with voltagedrops along the leads, especially in applications likeelectrochemiluminescence that often require relatively high currents.The problems are often greatest when using electrodes comprising thinlayers of only moderately conductive materials such as carbon inks. Theproblem may be partially mitigated by use of multi-layer patternedelectrodes (where the conductivity of an exposed moderately conductivematerial such as a carbon ink is increased by printing it over a moreconductive material such as a silver ink) although this approachintroduces additional manufacturing steps. Alternatively, the problemmay be partially mitigated in systems having multiple assay electrodesby keeping the leads short (preferably, so that the resistance betweenthe electrode and the electrical contact is less than 500 ohms, morepreferably less than 300 ohms, most preferably less than 100 ohms) tominimize the voltage drop and by keeping the leads about the same lengthto make the voltage drop consistent from electrode to electrode.

In an assay module comprising multiple working electrodes, thevariability from electrode to electrode in the voltage drop across theelectrode leads is preferably smaller than the potential applied duringthe course of an assay measurement so that this variability has minimaleffect on the variability of the measurements. In especially preferredembodiments, the variability in voltage drop across the leads is lessthan 20% of the potential applied during the course of an assaymeasurement, more preferably less than 10% or most preferably less than2%. Alternatively, the uniformity in leads can be described in terms ofthe variation in resistance across the leads which is preferably lessthan 50 ohms, more preferably less than 10 ohms, most preferably lessthan 1 ohm.

Where the arrangement of the electrodes and/or contacts makes itdifficult to keep the leads a uniform length, the matching of leadresistances can be accomplished by geometrically matching thelength-to-width ratio of each electrode lead (assuming consistent printthickness). This length-to-width ratio is referred to hereinafter as the“number of squares”. Typically, for a preferred cartridge-basedconfiguration using screen printed carbon inks, the electrode leads areon the order of 4 to 5 squares. Commercially available inks typicallyhave ink resistances that are specified in resistance per square perthickness (e.g., ohms/square/mil) and can vary widely depending on theink selected. In a particularly preferred embodiment, a carbon ink isused that possesses an ink resistance that measures approximately 15ohms/square/mil. The total resistance measured from end-to-end across alead for one preferred embodiment is typically on the order of 450 ohmsfor a configuration utilizing a 5 squares lead.

FIG. 2 depicts one preferred embodiment of an addressable electrodearray for generating ECL that can be incorporated into a cartridge-basedform factor having the requisite provisioning for sample/reagentmixing/delivery. As illustrated, contacts 205 and leads 210 are used toallow electrodes 215 in the addressable electrode array to be controlledby a control unit (not shown) adapted to contact, or mate, with thecartridge. Since the resistance across leads 210 represents a largefraction of the total cell resistance during an assay measurement, it ispreferable to match the resistance across each lead as closely aspossible. As shown in the figure, the length of the leads variesaccording to the positioning of the electrodes and contacts, however,the width is varied so that the length to width ratio of the leads iskept constant so as to provide a uniform lead resistance (the widths inthe figure are not to scale and have been exaggerated for emphasis).

Utilization of the electrode array for multiple purposes contributes toa miniaturized cartridge-based device since the need for additionalcomponents is obviated. According to another aspect of the presentinvention, the electrode array may advantageously also be used fordetecting the presence of fluid, for the detection of trapped air and/orfor the identification of sample type. Preferably, an impedancemeasurement may be used to monitor the state of the cell during thecartridge routine. The measurement may assess whether there is trappedair on or above an electrode during incubation and after the wash step.Additionally, the impedance measurement may also allow usage of theelectrode array to distinguish different sample types drawn into thecartridge, e.g., differentiate between samples of urine, saliva, serum,plasma, or whole blood, and make any necessary adjustments that may beneeded.

The advantages associated with utilizing the electrode array to monitorcartridge operations by performing impedance measurements can be manyfold. In particular, use of the electrode array in this manner affords anon-destructive measurement to be made since application of low voltageDC or, preferably, AC waveforms can be carried out with no effect on thesubsequent ECL measurement. Also, the impedance measurement performed bythe electrode array is relatively fast compared to other cartridgeoperations. Still further, the impedance measurement performed by theelectrode array is very precise and can preferably be used inconjunction with other sensors; e.g., pressure, optical, etc.

At low voltages, the electrodes located in the region where detection isto be made, i.e., the read chamber, behave like a series RC circuit.This has proven to be a suitable model for the development of a failsafe mechanism to ascertain the presence of fluid, the presence of anunwanted bubble or to discriminate between sample specimen in types inthe read chamber. In practice, it has been observed that trapped air mayreside either on the electrode surface or in the solution bulk.According to the present invention, the location of the air with respectto the electrodes is important. According to one embodiment, aresistance measurement can be utilized to provide an indicator that issensitive to air trapped in the bulk solution and at theelectrode/solution interface. According to another embodiment, acapacitance measurement can be employed to provide an indicator that isprimarily sensitive to air trapped at the interface. In yet anotheralternative embodiment, the electrochemical current during an ECLmeasurement (e.g., the TPA oxidation current during ECL) may be used todetect trapped air during the ECL measurement, however, this measurementwould not provide information related to trapped air during the sampleentry and incubation phases and would not allow corrective steps to betaken before the ECL measurement.

With respect to using a capacitance measurement, the pertinentcapacitance is the double layer capacitance. Since the parallel platecapacitance is insignificant at frequencies below about 1 MHz, it ispreferably ignored. Each electrode has a double layer capacitance. It isnoted that the double layer capacitance is not a true capacitor, as itdoes exhibit a small frequency dependence. Advantageously thecapacitance is primarily affected by changes at the interface (e.g.,changes in the effective area of an electrode due to the trapping of anair bubble on the electrode surface), and not by the bulk; thecapacitance is therefore preferably used to detect air bubbles at theelectrode/solution interfaces. Preferably, the capacitance measurementuses an AC voltage input with a frequency between 10-40,000 Hz, morepreferably between 20-2000 Hz, more preferably between 50-400 Hz, mostpreferably around 200 Hz. Other factors besides trapped air, e.g.,errors in the printing of the electrodes, may change the effective areaof an electrode and thus the measured capacitance. The measurement ofcapacitance can be used to check for these factors as well as forbubbles and can be used to trigger error flags if the capacitance valuesfall out of an acceptable range or, alternatively, to allow fornormalization of the reported ECL signal to compensate for the actualelectrode area.

With respect to using a resistance measurement, the pertinentresistances are the solution and lead resistances. It has been observedthat the solution resistance will have a small frequency dependence. Theresistance is affected by changes in the bulk solution (e.g., by bubblesinterfering with the flow of current through bulk solution) and changesat the electrode/solution interface (e.g., trapped air at the interfacehas the affect of reducing the effective electrode area and thereforeincreasing the resistance). The solution resistance can also be expectedto be very sensitive to the nature of the solution in contact with theelectrodes and can also be used to identify the sample.

The resistive (in-phase) and capacitive (out-of phase) components of theimpedance may be measured simultaneously using conventional impedanceanalyzing circuitry, preferably using a voltage waveform having afrequency at which both components have a significant effect on theimpedance and/or a voltage waveform having a plurality of frequenciescomprising at least one frequency where the resistance is a significantcomponent of the impedance and at least one frequency where thecapacitance is a significant component of the impedance. Alternatively,the resistive and capacitive components may be measured separately,preferably at frequencies that maximize the effect of the componentbeing measured. For example, at high frequencies the effect of surfacecapacitance is minimized and the impedance is primarily due to solutionresistance. In one embodiment of the invention, the solution resistanceis measured by applying a voltage waveform having a frequency greaterthan 2000 Hz, more preferably between 2,000 and 100,000 Hz, mostpreferably around 20,000 Hz.

Sample matrix identification can be very important since certainbiochemical assays may have varied steps or different post-processingrequirements (e.g., the blood samples may be treated different thanplasma samples). Tables 3 and 4 list resistance and capacitance valuesacquired for five different matrices by applying low voltage ACexcitation to electrodes within an experimental cartridge. The electrodearray comprised screen printed carbon ink electrodes, the exposedsurface of which were defined by a patterned dielectric layer printedover the carbon ink. The impedance measurements were taken at 25 degreesC. using an excitation voltage equal to 0.010 V rms at the frequenciesindicated in the tables. For capacitance measurements, since it isdesirable to use a frequency where all (or nearly all) of the voltagedrop occurs across the capacitive element, a frequency of 200 Hz wasutilized as this was found to result in greater than 95% of the voltagedrop to occur across the double layer capacitance; i.e., the solutionlosses were almost negligible. Resistance and capacitance werecalculated using a series RC model.

As can be seen in Tables 3 and 4, the capacitance varied little betweenthe different sample matrices, however, the resistances showed muchgreater variation among the matrices.

TABLE 3 Sample Discrimination Using Capacitance Measurements (phaseangles 76 to 82 degrees). Matrix Capacitance, uF at 200 Hz assay buffer0.023 saline 0.021 serum 0.019 plasma 0.018 blood 0.020

TABLE 4 Sample Discrimination Using Resistance Measurements (includes700 ohms of lead resistance; phase angles 12 to 16 degrees) MatrixResistance, ohms at 20,000 Hz assay buffer 2516 saline 3722 serum 3996plasma 4158 blood 7039

In certain preferred embodiments the electrochemical current measuredduring the induction of ECL, may be used to detect the presence oftrapped air over an electrode since trapped air may cause a significantdecrease in the electrochemical current (e.g., current from TPAoxidation during ECL). FIG. 5 depicts an image of ECL emitted from anelectrode array. One of the electrodes has a small dark spot 500 due thepresence of a small air bubble on the electrode surface. Even such asmall bubble gave a detectable change in the electrochemical currentmeasured at that electrode during the ECL experiment; the current in thepresence of the air bubble (178 uA) was significantly different (by 5%)than the average of the current at the other electrodes (187 uA). Otherfactors besides trapped air, e.g., errors in the printing of theelectrodes, may change the effective area of an electrode and thus themeasured current. The measurement of current during ECL can be used tocheck for these factors as well as for bubbles and can be used totrigger error flags if the current values fall out of an acceptablerange or, alternatively, to allow for normalization of the reported ECLsignal to compensate for the actual electrode area.

The bubble detection methods described above can also be employed todetect the presence of fluids, the presence of bubbles in fluids and/oridentify classes of samples in compartments in an assay cartridgeoutside the detection flow cells. For example, certain preferredembodiments of assay cartridges comprise fluid inlet and/or outlet linesfor introducing and removing fluids from the cartridge flow cells,wherein these inlet and/or outlet lines comprise fluid detectionelectrodes for detecting the presence of fluid, the presence of airbubbles in fluids and/or for identifying samples. These fluid detectionelectrodes may have independent electrode leads and contacts. So as toreduce the number of electrical contacts to the cartridge, these fluiddetection electrodes, preferably, comprise exposed surfaces of the leadsto assay electrodes (e.g., assay electrodes in the assay cartridge flowcells). In this arrangement, it is further preferred that the exposedleads in a given fluid volume (e.g., an inlet line or outlet line) donot comprise leads from two electrodes that will be fired together in anassay measurement (e.g., used as a working electrode counter electrodepair in an ECL measurement). In this fashion it is ensured that theassay measurements are not affected by low resistance current pathsbetween exposed leads.

With reference to the simplified embodiment depicted in FIG. 4, use ofthe impedance sensors 425 for detection of fluid presence and/ordiscrimination within the fluid input line 450 will now be discussed.Impedance sensors 425 are regions of electrically conductive surfaces onthe electrode leads between electrodes 401-407 and electrode contacts420. The electrically conductive surfaces are, preferably, exposed viaapertures in a patterned dielectric layer that is patterned over theelectrode leads. As fluid is directed into and through the fluid inputline 450 (e.g., by use of pumps, valves, capillary flow, and the like),the impedance sensors 425 may be activated by a controller (not shown)that applies interrogation potentials between sensor pairs to detectand/or discriminate the fluid (the interrogation potentials beingpreferably lower than those required to induce ECL at the assayelectrodes). The position of bubbles or fluids in the input line can bedetermined by sequentially measuring the impedance between differentsensor pairs and comparing the values. The sensors are on alternatingelectrode leads so that when adjacent electrodes are fired during, e.g.,an ECL measurement, the potential across the assay electrodes is notshort circuited by current between sensors.

According to another aspect of the present invention, the electrodesurfaces are coated with assay reagents such as antibodies or otherspecific binding reagents by dispensing solutions comprising thereagents to one or more appropriate locations on the electrode array,i.e., the capture surfaces. Preferably, the assay reagents collect onthe surface (e.g., via the formation of covalent bonds, non-specificadsorption or specific binding interactions) to form an immobilizedlayer on the electrode. In a preferred embodiment, accurate volumedelivery to a specified location results in complete coverage of onlythe desired electrode surface and/or a desired portion thereof. Accuratevolume delivery to a specified location can be readily accomplished withcommercially available dispensing equipment; e.g., commerciallyavailable equipment from BioDot.

Attaining complete coverage of a pre-defined region on a surface (e.g.,an assay electrode) via localized deposition of a liquid (e.g., an assayreagent or a liquid comprising an assay reagent) can be difficult toachieve if the advancing contact angle of the liquid on the surface ishigh, thereby inhibiting spreading of the liquid on the surface (as hasbeen observed for surfactant-free aqueous solutions on untreated carbonink electrodes). Spreading can be accelerated by chemically modifyingthe surface to make it more wettable or by adding surfactants to theliquid, however, in many circumstances it is undesirable to change thephysical properties of the surface or liquid. Alternatively, we havefound that excellent and well controlled spreading of liquids can beachieved on surfaces, such as carbon ink electrodes, having high contactangle hysteresis (i.e., large differences in the advancing andretreating contact angle of the liquid on the surface, preferablydifferences greater than 10 degrees, more preferably greater than 30degrees, more preferably greater than 50 degrees, most preferablygreater than 70 degrees) by using impact-driven fluid spreading. Suchresults can be achieved without surface modification or the use ofsurfactants. Fluid is deposited (preferably, using a fluidmicro-dispenser such as a micro-pipette, micro-syringe, solenoid valvecontrolled micro-dispenser, piezo-driven dispenser, ink-jet printer,bubble jet printer, etc.) on the surface at high velocity (preferablygreater than 200 cm/s, more preferably greater than 500 cm/s, mostpreferably greater than 800 cm/s) so as to drive spreading of the liquidover the surface, despite the high advancing contact angle, to a sizedictated by the volume and velocity of the dispensed fluid. The lowretreating contact angle prevents significant retraction of the fluidonce it has spread. Using the impact-driven spreading technique, it ispossible to coat, with a predetermined volume of liquid, regions of asurface that are considerably larger (preferably, by at least a factorof 1.2, more preferably by at least a factor of two, even morepreferably by at least a factor of 5) than the steady state spreadingarea of the predetermined volume of liquid on the surface (i.e., thearea over which a drop having that volume spreads when touched to thesurface at a velocity approaching zero).

Preferably, the region to be coated is defined by a physical boundarythat acts as a barrier to confine the deposited fluid to the pre-definedregion (e.g., a surrounding ledge or depression, a boundary formed ofpatterned materials deposited or printed on the surface, and/or aboundary formed via an interface with a surrounding region that variesin a physical property such as wettability). More preferably, the liquidhas a higher receding contact angle on the surrounding region than onthe pre-defined region (preferably, the difference is greater than 10degree, more preferably greater than 30 degrees, most preferably greaterthan 50 degrees). Even more preferably, the surrounding region alsoexhibits a low contact angle hysteresis for the liquid (preferably, lessthan 20 degrees, most preferably, less than 10 degrees). By using asurrounding region having high receding contact angle and/or lowhysteresis, the tolerance for imprecision in deposition velocity orspreading rate becomes much improved. In a preferred deposition method,a small volume of reagent is dispensed onto the pre-defined region withsufficient velocity to spread across the pre-defined region and slightlyonto the surrounding region, the liquid then retracts off thesurrounding region (due to its high receding contact angle) but does notretract smaller than the size of the pre-defined area (due to its lowreceding contact angle). In especially preferred embodiments of theinvention the pre-defined area is an exposed area of an electrode(preferably, a carbon ink electrode) and the surrounding region isprovided by a dielectric ink patterned on the electrode.

FIG. 8 illustrates typical observed contacts angles of 250 nL drops ofwater deposited using a solenoid valve-controlled micro-dispenser(Bio-Dot Microdispensor, Bio-Dot Inc.) on a preferred dielectric ink anda preferred carbon ink. The figure plots the contact angle as a functionof the velocity of fluid as it leaves the tip of the dispenser. At lowvelocity, the observed contact angle is close to the advancing contactangle of water on the surface. As the velocity increases, impact-drivenspreading causes the liquid to spread over a greater area and theobserved contact angle decreases. At the high velocities, the observedcontact angle becomes relatively independent of velocity as itapproaches the receding contact angle of the liquid on the surface, thereceding contact angle being the lowest contact angle the liquid canhave on the surface (a lower contact angle would cause the drop torecede till it achieves the receding contact angle).

As described above, assay reagents such as antibodies or other specificbinding reagents may be patterned by depositing (e.g., via impact drivenspreading) solutions comprising the reagents on pre-defined locations ona surface (e.g., an electrode surface, preferably a carbon ink electrodesurface) and allowing the reagents to become immobilized on the surface(e.g., via covalent bonds, non-specific interactions and/or specificbinding interactions). Preferably, the region to be coated is defined bya physical boundary that acts as a barrier to confine the depositedfluid to the pre-defined region (e.g., a surrounding ledge ordepression, a boundary formed of patterned materials deposited orprinted on the surface, and/or a boundary formed via an interface with asurrounding region that varies in a physical property such aswettability) so as to form a fluid containment region.

In certain preferred embodiments, antibodies or other binding reagents(preferably proteinaceous binding reagents) are immobilized on carbonink electrodes by non-specific adsorption. It may be advantageous toallow the assay reagent solution to dry on the electrode during theimmobilization procedure. Preferably, the immobilization procedurefurther comprises blocking un-coated sites on the surface with ablocking agent such as a protein solution (e.g., solutions of BSA orcasein), washing the surface with a wash solution (preferably a bufferedsolution comprising surfactants, blocking agents, and/or proteinstabilizers such as sugars) and/or drying the surface.

In a preferred immobilization procedure of the invention, imprecisiondue to variations in the ability of different assay reagents to adsorbon a surface such as a carbon ink electrode are reduced by immobilizingvia a specific binding interaction involving a first and second bindingpartner. Such an immobilization technique is less likely to be affectedby small variations in the properties of the surface. By way of example,antibodies may be patterned by patterned deposition of antibodysolutions (the first binding partner) on a surface coated with anantibody binding reagent (the second binding partner, e.g., ananti-species antibody, protein A, protein G, protein L, etc.).Alternatively, assay reagents labeled with the first binding partner(preferably, biotin) may be patterned by patterned deposition of theassay reagents on a surface coated with the second binding partner(preferably, anti-biotin, streptavidin, or, more preferably, avidin).Most preferably, the second binding partner is deposited in the samepattern as the assay reagents. By analogy, the method can be adapted touse any of a variety of known first binding partner—second bindingpartner pairs including, but not limited to, hapten-antibody, nucleicacid—complementary nucleic acid, receptor-ligand, metal-metal ligand,sugar-lectin, boronic acid—diol, etc.

Accordingly, one embodiment of an immobilization method of the inventioncomprises forming an assay domain comprising an assay reagent by: i)treating a predefined region of a surface (preferably, a carbon inkelectrode surface) with a solution comprising a second binding partnerso as to form an adsorbed capture layer (or, alternatively, a covalentlybound layer) of said second binding partner (preferably, avidin) withinthe predefined region of said surface; (ii) treating the capture layerin the pre-defined region with a solution comprising the assay reagent,wherein the assay reagent is linked to or comprises a first bindingpartner (preferably, an assay reagent that is labeled with biotin) thatbinds the second binding partner. Preferably, a micro-dispensingtechnique is used to pattern the second binding partner and/or the assayreagent into the pre-defined region (more preferably both arepatterned). More preferably, the pre-defined region is defined by aboundary (preferably defined by a dielectric layer patterned on thesurface) adapted to confine small volumes of fluid to the pre-definedregion.

The treating steps may comprise allowing the solutions to dry on thepre-determined regions. Between binding the second binding partner andbinding the assay reagent, it may be advantageous to wash the surfacewith one or more wash solutions to remove excess unbound second bindingpartner. The wash solutions, preferably, comprise surfactant and/orblocking agents. After immobilizing the assay reagent, it may beadvantageous to wash the surface with one or more wash solutions toremove unbound assay reagent. The wash solutions, preferably, comprisesurfactants, blocking agents and/or protein stabilizers such as sugars.Useful blocking agents include standard blocking agents of the art (BSA,casein, etc.) but also include blocking reagents comprising the firstbinding partner (for example, free biotin) so as to block free bindingsites on the immobilized layer of the second binding reagent. The washsteps may employ the wash techniques of the invention that employconcentric tubes for adding and removing wash solution. The surfaces areoptionally dried after preparation for long term storage.

Preferably, the amounts of the second binding reagent and assay reagentapplied to the pre-defined region are equal to or less than thatrequired to saturate the surface. By choosing amounts roughly equal tothe amounts required to saturate the surface, it may be possible tominimize both the amount of excess unbound reagent and the amount ofunbound sites and thus avoid the need for washing or blocking steps. Inan alternative embodiment, the amount of the assay reagent is kept belowthe amount of available binding sites in the capture layer to ensurethat the binding capacity is determined by the amount of assay reagentadded and not by amount of immobilized second binding partner (thusreducing the effect of variability in the efficiency of, e.g., theadsorption of the second binding partner).

The method may be applied to forming a plurality of assay domainscomprising assay reagents immobilized in a plurality of pre-definedregions. In this case, the method is simply repeated for each of thepre-defined regions. Preferably, at least two of the assay domainscomprise assay reagents that differ in selectivity for analytes ofinterest. When forming a plurality of assay domains, it is particularlyadvantageous to block the final product with a blocking reagentcomprising the first binding partner (but not the analyte specificcomponents of the assay reagent) to block excess binding sites onimmobilized second binding partners; this procedure prevents assaycross-talk due to excess assay reagent on one pre-defined regiondiffusing and binding, via first binding partner-second binding partnerinteractions, to a different assay domain. For example, after using thetwo step procedure of binding avidin and then a biotin-labeled antibody,the surface may be blocked with free biotin. Alternatively, after usinga two step procedure of binding Protein A (or other Fc binding receptor)and then an antibody against an analyte of interest, the surface may beblocked by using a different antibody or, more preferably, an Fcfragment of an antibody.

It has been observed that in some cases assay reagents adsorbed on asurface such as a carbon ink may, over time, slowly dissociate from thesurface. This dissociation leads to the presence of free assay reagentsthat may interfere with assays that employ the adsorbed assay reagents.This dissociation may be greatly slowed by cross-linking the adsorbedassay reagents so that the immobilized species are greater in molecularweight and have more points of contact with the surface. Accordingly, inthe immobilization methods described above, the second binding partneris, preferably, cross-linked to minimize dissociation of the reagentduring surface preparation and/or storage. The cross-linking may becarried out via covalent cross-linking using standard chemicalcross-linking agents. Alternatively, the cross-linking is carried outusing specific binding interactions. In a preferred embodiment of theinvention, the second binding partner is polyvalent (i.e., has multiplebinding sites for the first binding partner) and is cross-linked bycombining it with a cross-linking reagent that is either a polyvalentfirst binding partner or a molecule which comprises multiple firstbinding partners. In this embodiment, the amount of the cross-linkingagent is selected so as to provide a beneficial amount of cross-linkswithout saturating all the available binding sites on the second bindingpartners. The cross-links may be formed after the second binding partneris immobilized but are, preferably, formed in solution prior toimmobilization. Advantageously, we have found that this cross-linkingprocedure not only acts to form a more stable surface but also increasesthe number of available binding sites on the surface (i.e., the bindingcapacity of the surface) by allowing the immobilization of more than apacked monolayer of the second binding partner (e.g., by extension ofthe polymerized second binding partner into solution).

By way of example, avidin (a tetrameric binding protein having fourbinding sites for biotin) is cross-linked to form poly-avidin by theaddition of a small quantity of biotin-labeled cross-linking agent (forexample, a protein such as BSA) having multiple biotin labels perprotein molecule. Poly-avidin is then immobilized and used as a capturesurface for immobilizing a biotin-labeled assay reagent, e.g., using theimmobilization methods described above. The amount of biotin-protein isselected to allow cross-linking while leaving sufficient biotin bindingsites available so that the immobilized poly-avidin can be used tocapture a biotin-labeled first binding reagent (e.g., a biotin-labeledantibody). Preferably, the biotin-labeled cross-linking agent comprisesat least two, more preferably, at least four, or more preferably, atleast eight biotins per molecule. Preferably, the number of molarequivalents of cross-linking agent per mole of avidin is between 0.01and 4, more preferably, between 0.01 and 1, even more preferably between0.01 and 0.25, even more preferably between 0.05 and 0.25 and mostpreferably between 0.05 and 0.10. The concentration of avidin used forimmobilization was preferably between 50-1000 ug/mL, more preferablybetween 100-800 ug/mL and most preferably around 400 ug/mL. By analogy,avidin may be replaced in these methods by other poly-valentbiotin-specific receptors such as streptavidin.

Experiments were conducted to demonstrate the benefit of usingpoly-avidin capture layers on carbon ink electrodes and/or the two-stepimmobilization procedures of the invention. These experiments usedscreen printed carbon ink electrodes that were patterned on a plasticsubstrate. The working electrodes had an exposed circular area of about3 mm² that was defined by a patterned dielectric layer that was screenprinted over the carbon ink electrodes. The substrate also comprised atleast one additional carbon ink electrode for use as a counterelectrode. Reagents were immobilized by depositing (using a Bio-Dotdispenser) small volumes (200-300 nL) of a solution comprising thereagent onto the exposed electrode area (the solution being confined tothe exposed electrode area by the dielectric layer) and allowing thesolution to dry on the electrode. Poly-avidin was prepared by combiningthe appropriate amounts of avidin and biotin-BSA and incubating for 15minutes. After the immobilization and/or washing steps (as describedbelow), the substrate was either mated with a multi-well plate top so asto form the bottom surface of a well of multi-well plate or it was matedusing a gasket made of double stick tape to a plastic sheet so as toform the bottom surface of a flow cell of an assay cartridge. Theelectrode surfaces were contacted with a buffered solution comprisingtripropylamine (MSD Assay Buffer, MSD) by adding the buffer to a well ofa multi-well plate or by introducing the buffer into the flow cell. ECLwas induced by applying a voltage between the working and counterelectrode (a ramp of 2-5 V over 3 seconds). ECL was measured by takingan image of the substrate using a cooled CCD camera.

Electrodes were coated with either avidin (by treating with 200 nL of a75 ug/mL solution of avidin) or with poly-avidin (by treating with 200nL of a solution containing 75 ug/mL avidin and 3.1 ug/mL biotin-labeledBSA and allowing the solutions to dry overnight; the BSA being labeledwith a 4-fold excess of biotin-LC-sulfo NHS ester and having an expectedratio of biotins per BSA of roughly 2-3). The substrates were washedwith water and the electrodes were then treated with 300 nL of asolution containing 100 ug/mL of an biotin-labeled anti-TSH antibody.The electrodes were washed with water, assembled into a cartridge intowhich was introduced a solution containing 20 uIU/mL of TSH and 12 ug/mLof an anti-TSH antibody that was labeled with a Sulfo-TAG NHS ester(MSD), an electrochemiluminescent label. The cartridge was incubated for8 minutes to allow the binding reactions to occur, the substrate wasthen washed by passing MSD Assay Buffer into the flow cell and ECL wasmeasured. The average emitted electrochemiluminescence intensity fromthe poly-avidin treated electrode (1652 units) was approximately threetimes that from the avidin treated electrode (602 units). Without beingbound by theory, it is believed that the higher signal on thepoly-avidin electrode represents an increased number of binding sites onthe poly-avidin treated electrode and/or a reduction in the amount ofavidin that washes off the poly-avidin electrode and adsorbs on othersurfaces of the cartridge (thus competing with binding sites on theelectrode).

In a similar experiment, the direct adsorption of anti-TSH antibody (bytreatment of the electrode with a 100 ug/mL solution of an anti-TSHantibody) was compared to immobilization via a poly-avidin layer (asdescribed above except that the poly-avidin solution contained 400 ug/mLavidin and 25 ug/mL biotin-BSA and the biotin-labeled anti-TSH was at aconcentration of 100 ug/mL). The results showed that signal obtainedusing immobilization via poly-avidin (2207) was roughly twice thatobtained using direct adsorption (1264). In addition, two stepimmobilization protocol was found to provide more precise results; thecoefficients of variation (CVs) were three times lower when the two stepmethod was employed.

The poly-avidin layers were further characterized by using avidin thatwas labeled with an electrochemiluminescent label (on average 0.3Sulfo-TAG NHS labels per protein). The electrodes were treated with oneof three solutions: (i) 75 ug/mL avidin, (ii) 75 ug/mL avidin and 25ug/mL BSA or (iii) 75 ug/mL avidin and 25 ug/mL biotin-BSA. All thesolutions contained 0.0035% Triton X-100. The electrodes were washedwith water, immersed in MSD Assay Buffer and ECL was measured. Theelectrode treated with all the components of poly-avidin (avidin andbiotin-BSA) gave an ECL signal (150981) that was roughly twice thatobserved for avidin alone (85235) or avidin with unlabeled BSA (65570),demonstrating that cross-linking was required for the improvedperformance of poly-avidin. It was also observed that the intensity ofECL was much more evenly distributed across the electrode for thepoly-avidin electrodes than for the other electrodes.

In a different experiment the labeled and immobilized avidin orpoly-avidin layers were i) not washed or ii) exposed to a solutioncontaining BSA for 2 hours and then extensively washed with phosphatebuffered saline. In this experiment, the avidin concentration was 0.5mg/mL, the ratio of avidin to biotin-BSA was 16:1 and the labeled avidinwas mixed with unlabeled avidin (at a 1:1100 ratio) to reduce theoverall signals. The experiment was carried out on both non-treatedelectrodes and electrodes that were treated with an oxygen plasma. Thetable below shows that the use of poly-avidin substantially reduced theloss of avidin from the surface after extensive washes and exposure toprotein-containing solutions.

Unmodified Electrodes Plasma-Treated Electrodes Avidin Poly-AvidinAvidin Poly-Avidin Signal % Left Signal % Left Signal % Left Signal %Left No Wash 21,107 26,618 10,871 18,512 Wash 9,545 45 18,845 71 3,33231 14,024 76

After immobilizing assay reagents on surfaces for use in solid phaseassays (e.g., by applying solutions comprising the assay reagents to thesurfaces, most preferably, by patterned depositions of these solutionsto form an array of assay domains comprising the assay reagents), assayperformance is often improved by washing the assay electrodes to removeunbound assay reagents. This washing step is particularly important whenunbound assay reagent may interfere with an assay (e.g., unboundantibodies may interfere by competing with the capture of analytes toantibodies on the surface). Preferably, this washing step is carried outusing a procedure that minimizes the ability of unbound reagents toadsorb in other undesirable locations. For example, after immobilizationof an antibody on an assay domain on an electrode in an assay module,the washing step will preferably minimize the adsorption of unboundantibody to non-electrode surface (antibody adsorbed on non-electrodesurfaces interfering with binding assays by competing for the binding ofanalyte with antibody immobilized on the electrode). Even moreimportantly, in array type measurements involving a plurality of assaydomains specific for different analytes of interest, the washing stepshould minimize the diffusion of an unbound assay reagent from one assaydomain and its adsorption on a different assay domain (this processleading to assay cross-talk).

We have found that we can prevent the undesired adsorption of assayreagents outside pre-defined locations by localized washing of assaydomains using a concentric tube dispense/aspirate fixture. FIGS. 7 a and7 b depict one embodiment wherein a washing fixture was constructed thatconsists of a single concentric tube structure which may be used to washa single assay domain in an assay module or to sequentially washmultiple assays domains in an assay module by positioned the concentrictube structure over each assay electrode. It should be understood,however that the invention is not limited to a single concentric tubedevice but can, preferably, employ an array of concentric tubes,preferably, arranged in the same pattern and spacing as the assaydomains. Preferably, wash fluid is dispensed through inner tube 705 andaspirated through outer tube 710. In operation, as the fluid transitionsfrom the inner tube to the outer, it preferably passes over the assaydomain surface, washing the assay domain in an area confined by thediameter of the outer tube. The figure shows the concentric tube beingused to wash a carbon ink electrode 720 patterned on substrate 730, theexposed surface of electrode 720 being defined by patterned dielectriclayer 725 which acts as a boundary to form a fluid containment region onelectrode 720. By analogy, the concentric tubes may be used to washassay domains on a variety of other surfaces, the assay domains beingpreferably but not necessarily defined by a fluid boundary. The tubesare preferably configured so that the outer tube removes fluid with ahigh enough efficiency so as to prevent the spread of fluid to regionsoutside the domain being washed. In alternate embodiments, the functionsof the inner and outer tubes may also be reversed such that the washfluid is dispensed through the outer tube, and aspirated up the centervia the inner tube. These arrangements of tubes prevent unbound assayreagents on the assay domains from contacting other surfaces of theassay module.

In another alternate embodiment, a tube structure having threeconcentric tubes is used to pattern and wash assay reagents on assaydomains. A first tube (preferably the inner tube) is used tomicrodispense assay reagents on an assay domain. This tube is preferablylinked to a low volume fluid dispensing controller such as amicrosyringe (optionally, having a solenoid valve flow controller) orpiezoelectric dispenser. The second tube (preferably the middle tube) isused to dispense bulk washing reagents on the assay domain. The thirdtube (preferably the outer tube) is used to aspirate excess assayreagent and/or to wash reagents from the assay domain. Using thisarrangement, a single device may be used to dispense assay reagents ontoan assay domain (e.g., so as to cause localized immobilization of theassay reagent on the assay domain) and to wash excess assay reagent fromthe assay domain, these operations occurring without contamination ofadjacent surfaces with the assay reagent. Optionally, an array of thesedevices is used to pattern and wash an array of assay domains.

The invention relates in part to assay cartridges. An assay cartridge ofthe invention incorporates one or more fluidic components such ascompartments, wells, chambers, fluidic conduits, fluid ports/vents,valves, and the like and/or one or more detection components such aselectrodes, electrode contacts, sensors (e.g., electrochemical sensors,fluid sensors, mass sensors, optical sensors, capacitive sensors,impedance sensors, optical waveguides, etc.), detection windows (e.g.,windows configured to allow optical measurements on samples in thecartridge such as measurements of absorbance, light scattering, lightrefraction, light reflection, fluorescence, phosphorescence,chemiluminescence, electrochemiluminescence, etc), and the like. Acartridge may also comprise reagents for carrying out an assay such asbinding reagents, detectable labels, sample processing reagents, washsolutions, buffers, etc. The reagents may be present in liquid form,solid form and/or immobilized on the surface of solid phase supportspresent in the cartridge. Certain preferred embodiments of theinvention, comprise detection chambers having the electrode arraysand/or binding domains as described above (e.g., the electrode arraysdescribed in FIGS. 1-4).

The fluidic components are preferably designed and incorporated into thecartridge body to form the fluidic network using certain predefineddesign guidelines. The design guidelines for each component can bedependent upon one or more factors such as, e.g., cartridge body design(i.e., single-piece body, multiple piece body, modular body, single readchamber, multiple read chamber, and the like), manufacturing process(e.g., injection molding, blow molding, hot stamping, casting,machining, etc.), materials (e.g., acrylic, PVDF, PET, polystyrene,polypropylene and the like), assay requirements (e.g., binding assay,competitive binding assay, single step assay, two-step assay, etc.),functional requirements (e.g., sample size, assay reagent volumes,detection technology, time-to-result, incubation, heating,mixing/agitating), safety/handling requirements (e.g., self-containment,regulatory approval, ease of use, etc.), and/or the like.

The skilled practioner will be able to readily select materials suitablefor the fabrication of the cartridges of the invention. Suitablematerials include glass, ceramics, metals and/or plastics such asacrylic polymers (such as Lucite), acetal resins (such as Delrin),polyvinylidene fluoride (PVDF), polyethylene terephthalate (PET),polytetrafluoroethylene (e.g., Teflon), polystyrene, polypropylene, ABS,PEEK and the like. Preferably, the materials are inert to anysolutions/reagents that will contact them during use or storage of thecartridge. In certain preferred embodiments, at least some portion ofthe cartridge is fabricated from transparent and/or translucentmaterials such as glass or acrylic polymer to provide windows that allowoptical interrogation of fluids or surfaces inside the cartridge, e.g.,for analysis of compositions within detection chambers of the cartridgeor for monitoring and controlling the movement of liquids through thefluidic networks defined within the cartridge.

One preferred embodiment of the invention is a cartridge that includesone or more sample chambers, one or more detection chambers (preferably,detection chambers adapted for use in ECL measurements as describedabove) and one or more waste chambers. The chambers are connected inseries by fluid conduits so that a sample introduced into a samplechamber can be delivered into one or more detection chambers foranalysis and then passed into one or more waste chambers for disposal.Preferably, this cartridge also includes one or more reagent chambersfor storing liquid reagents, the reagent chambers connected via conduitsto the other components so as to allow the introduction of the liquidreagents into specified sample or detection chambers. The cartridge mayalso include vent ports in fluidic communication with the sample,detection and/or waste chambers (directly or through vent conduits) soas to allow the equilibration of fluid in the chambers with theatmosphere or to allow for the directed movement of fluid into or out ofa specified chamber by the application of positive or negative pressure.

In an alternative embodiment, a sample chamber and a waste chamber areboth arranged upstream from a detection chamber having first and secondinlet/outlet conduits (preferably, a detection chamber having anelongated shape, the inlet/outlet conduits being arranged at or near theopposite ends of the elongated dimension). The cartridge is configuredto allow the introduction of sample into the detection chamber via thefirst inlet/outlet conduit and then the reversal of flow to direct thesample fluid back out the first inlet/outlet conduit and to the wastechamber. Preferably, a reagent chamber is located downstream of thedetection chamber and the cartridge is configured to allow introductionof the reagent to the detection chamber via the second inlet/outletconduit (i.e., in “reverse flow” relative to the introduction ofsample). This arrangement is particularly well suited to measurementsthat suffer from strong sample interference, the reverse flow beingespecially efficient at washing residual sample from the detectionchamber. This embodiment is especially useful in ECL-based assays formarkers (e.g., cell wall markers of gram positive bacteria) in samplescontaining a nitrous acid-containing extraction buffer (see, e.g., theextraction methods and reagents disclosed in U.S. Provisional PatentApplication 60/436,591, filed Dec. 26, 2002, entitled MethodsCompositions and Kits for Biomarker Extraction, hereby incorporated byreference). One preferred embodiment of the invention uses a cartridgeconfigured with a reverse flow wash to conduct an ECL binding assay fora panel of upper respiratory pathogens including streptococcal speciesand optionally other pathogens such as influenza A and B and RSV(preferably by employing an array of antibodies against markers of thepathogens, the array preferably being formed on one or more electrodes,most preferably an electrode array as described above and in FIGS. 1-4).

The reverse flow wash significantly reduces the detrimental effects ofnitrous acid on ECL measurements. In preferred embodiments, the washingefficiency is such that the fraction of sample (or reagent) left in adetection chamber after a wash is less than 1/1000; more preferably lessthan 1/10,000, even more preferably less than 1/100,000

The sample chamber is a chamber defined within a cartridge that isadapted for receiving a sample to be analyzed in the cartridge. Thesample chamber includes a sample introduction port for introducingsample into the chamber. The port is preferably an opening in thecartridge that provides access to the sample chamber. Alternatively, theport may be a membrane or septa through which a sample may be injectedinto the sample chamber, e.g., through the use of a needle or cannula.Preferably, the cartridge also includes a sealable closure for sealingthe sample introduction port and preventing leakage of the sample andpossible exposure of the user and/or associated instruments tobiohazards. Preferably the sealing/capping mechanism utilizes a hingedconfiguration so that the sample chamber is easily accessed and sealed.In particularly preferred embodiments the sealing/capping mechanismincorporates a flexible hinge, e.g., rubber, plastic or the like. Mostpreferably, the sample chamber is adapted and configured to receive amodular detachable insert that includes a cap for sealing the samplechamber. Use of a modular detachable insert within the sample chamberalso allows for independent selection of materials for the maincartridge body. In an alternative embodiment, sealing of the sampleintroduction port is achieved by applying an adhesive tape to the port.The sample chamber may contain dry reagents used in carrying out theassay that reconstitute on addition of a liquid sample. Optionally, thesample chamber contains an anti-foam agent to prevent foaming of thesample in the cartridge.

The sample chamber is connected to a sample conduit for transferringfluids from the sample chamber to other fluidic components in thecartridge. The sample chamber may also be connected to a vent portand/or a reagent chamber (e.g., through fluidic conduits). In apreferred configuration for receiving liquid samples, the sample chamberis connected to a sample conduit and a vent port. A cross-sectional viewof a preferred embodiment is shown in FIG. 27. Sample chamber 2710 hassample introduction port 2720 and is linked to sample conduit 2730 andsample vent port 2740 (through vent conduit 2750). Sample conduit 2730is advantageously arranged to intersect sample chamber 2710 at or nearthe bottom of the chamber (relative to the orientation of the cartridgeduring operation) so as to allow for efficient transfer of a largefraction of the sample volume without the introduction of bubbles. Ventconduit 2750 is advantageously arranged to intersect sample chamber 2710above sample conduit 2730 and at a height that is greater than theanticipated sample fill level height to avoid possible contamination ofthe instrument and/or escape of the sample fluid. Preferably, ventconduit 2750 has sufficient volume in the fluidic conduit so that asmall amount of sample fluid, e.g. as may be observed if the sample isfoamy or has bubbles, may enter the conduit without being pulled all theway to vent port 2740. In one embodiment, as depicted in FIG. 9, awell/trap 975 may be arranged within the fluidic conduit. In anotherembodiment, as depicted in FIG. 20, the fluidic conduit may beextended/lengthened, e.g., utilizing a serpentine configuration 2030.

Cap 2760 can be used to seal sample introduction port 2720 withoutpreventing the flow of air through vent conduit 2750. In FIG. 27, thefluidic compartments and conduits are formed by recesses (e.g.,channels) or holes in cartridge body 2770 and by cover layer 2780 whichis sealed against cartridge body 2770. Sample chamber 2710 has internalledge 2790. Vent conduit 2750 includes a vertical hole from the bottomof cartridge body 2770 to the top face of ledge 2790. This arrangementprovides for a simplified manufacturing process that is amenable toinjection molding or machining of the cartridge body; other arrangementsof the vent conduit will be readily apparent to the skilled artisan.

In one embodiment of the sample chamber, a separate vent port and ventconduit are omitted and the sample introduction port also provides avent port, e.g., the sample introduction port aperture also acts as avent port. The vent port may also be provided through the top of thesealing/capping mechanism by, e.g., incorporating a vent hole in the topsurface of the sealing/capping mechanism. An alternative embodiment mayemploy a scheme whereby the cartridge reader itself can include apiercing/venting mechanism that is adapted and configured to piercethrough the top surface of the flexible sealing/capping mechanism. In aparticularly preferred embodiment, the sealing/capping mechanism isadapted and configured to be self-sealing upon withdrawal/removal of thepiercing/venting mechanism, e.g., via the use of a septum preferablycomprising an elastomeric material. The advantage of a self-sealing capmechanism is that the sample cannot escape from the sample chamber oncethe piercing/venting mechanism has been removed.

The sample chamber may also include a filter for, e.g., removingparticulate matter that may be present within the sample itself or thatmay be present as a result of using a swab or the like to introducesample into the sample chamber. A preferable embodiment may employ afilter that not only removes any particulate matter but that is alsodesigned to separate red blood cells (RBC) from blood plasma; e.g.,where the particular assay/assay format requires blood plasma as thesample. Such a filter can be an integral cross-flow filter, in-linefilter or the like. Preferably, the filter is arranged at or near theentrance of the sample conduit.

In a preferred embodiment for extracting analytes from a solid matrix ora matrix that comprises solids (e.g., for extracting analytes from anabsorbent material (e.g., a cotton ball, piece of filter paper, etc.),an applicator stick, dirt, food, sludge, feces, tissue, etc.) the samplechamber is connected to a reagent chamber (e.g., via a reagent conduit)comprising an extraction reagent, e.g., an extraction reagent disclosedin U.S. Provisional Patent Application 60/436,591, filed Dec. 26, 2002,entitled Methods Compositions and Kits for Biomarker Extraction, herebyincorporated by reference. Applicator stick is used herein to refer to asample collection device comprising an elongated handle (preferably arod or rectangular prism) and a sample collection head (preferablycomprising an absorbant material or, alternatively, a scraping blade)configured to collect sample from a surface or biological tissue) andincludes sample collection swabs and tissue scrapers. The reagentconduit and sample conduit are, preferably, arranged to intersect thesample chamber at or near opposing ends of the chamber so that reagentintroduced through the reagent conduit is drawn through the samplebefore passing into the sample conduit. More preferably, the samplechamber has an elongated shape with the two conduits being arranged tointersect at or near the opposing ends of the length. The sample chambermay also include a filter, as described above, for removing solidmaterial. Extraction of analytes from solid materials and, inparticular, porous meshes such as may be found in swab heads may lead tothe introduction of bubbles and air gaps into the resulting fluidstream. Preferably, the sample chamber or the downstream fluidiccomponents (e.g., the sample conduit) include a bubble trap to removeair introduced during an extraction step.

FIG. 28 shows a cross-sectional view of one exemplary embodiment of asample chamber for extracting analyte from a solid or solid-containingmatrix. Elongated sample chamber 2810 has a sample introduction port2820 equipped with a sealable closure as described above. The samplechamber is shown holding an applicator stick, specifically swab 2830having absorbent swab head 2835. Reagent conduit 2840 and sample conduit2845 are arranged to intersect sample chamber 2810 on opposing sides ofswab head 2835 so that extraction reagent introduced through reagentconduit 2840 passes through swab head 2835 before entering sampleconduit 2845. Optionally, a filter element 2848, may be included toremove particulates from the extracted sample. Preferably, the width ofsample chamber 2810 in the region that surrounds the head of an insertedapplicator stick is less than two times (more preferably less than 1.5times, even more preferably less than 1.2 times, most preferably equalto or less than 1.0 times) the width of the widest region of theapplicator stick that needs to pass through that region during insertionof the applicator stick. Alternatively, the cross-sectional area ofsample chamber 2810 in the region that surrounds the head of an insertedapplicator stick is less than four times (more preferably, less than twotime, most preferably less than or equal to 1.0 times thecross-sectional area of the widest region of the applicator stick thatneeds to pass through that region. When used to extract sample fromporous compressible materials (e.g., a swab having a porous compressiblehead), the width of the sample chamber is selected so that the width isnarrow enough around the applicator stick head so that the materialfills most or all the width of the chamber (ensuring the most efficientflow of extraction buffer through the material) but wide enough so thatmaterial can be easily inserted without the need for excessive force andwithout causing leakage of fluid in the material onto the outsidesurfaces of the cartridge (optionally, both properties may be achievedby use of a chamber that, with respect to a seated applicator stick isnarrower in the region that surrounds the head than in the region thatsurrounds the shaft). In certain preferred embodiments, these propertiesare achieved while. Advantageously, sealing sample port 2820 preventsthe release of air from that end of sample chamber 2810 and prevents thewasteful flow of extraction reagent away from sample conduit 2845.Optionally, swab 2830 and/or chamber 2810 are designed so that swab 2830fits completely into chamber 2810. Alternatively (as shown), anapplicator stick is too long to fit in chamber 2810 (e.g., the length ofswab necessary to collect a mucous sample from the throat or nasalcavity may be too long to fit within the desired form factor of acartridge) but is cleaved (e.g., broken, fractured, cut or otherwisedetached) prior to or, preferably, after its introduction into chamber2810 so as to produce a shortened stick fragment comprising the samplecollection head. The shortened fragment is short enough to fit inchamber 2810 and allow closure 2825 to be sealed. In certainembodiments, the swab is designed to allow for easy detachment byhaving, e.g., a reversibly detachable head or by including a weak pointin the shaft that allows for facile fracture of the shaft.

One method of introducing an applicator stick such as swab 2830 tosample chamber 2810 comprises i) introducing it into chamber 2810; ii)cleaving the swab shaft to form a head segment (comprising the head) anda shaft segment and iii) sealing the head segment in chamber 2810 bysealing closure 2825. The method may further comprise iv) introducing anextraction reagent through reagent conduit 2840; v) extracting analytefrom swab head 2830 by passing extraction reagent through swab head 2835and vi) removing the extracted analyte through sample conduit 2845. Theextracted analyte may then be directed to a detection chamber foranalysis. In one preferred embodiment, the shaft is cleaved by applyinga force to the exposed end of the shaft of swab 2830 in a directionperpendicular to the length of chamber 2810 so as to break the shaft atan edge 2827 of chamber 2810 and allow removal of the part of the shaftthat extends out of the chamber. Preferably, swab head 2830 is seatedagainst the opposing end of chamber 2810 prior to cleaving the shaft.

In an especially preferred embodiment, the shaft of swab 2830 isconstructed to have weak point (shown as weak point 2837) so thatapplication of a force causes swab 2830 to reproducibly break at theweak point. Preferably, the swab shaft includes a stress/strainconcentration feature (notch, score, or the like), e.g., the weak pointis introduced by making the swab shaft narrower at the weak point or by“scoring” the shaft (i.e., cutting or etching one or more notches intothe shaft at the weak point). Preferably the notch forms a circuitaround the shaft so that the shaft may be broken in any direction. Sucha notch may be made by cutting a groove in the shaft (e.g., with a toolor a laser) while turning the applicator stick on a lathe. Mostpreferably, the weak point is located so that when the shaft is insertedinto chamber 2810 it is sufficiently near to edge 2827 so that asufficient force can be applied to break the shaft, but sufficientlyclose to head 2835 so that the closure 2825 can be sealed.

The sample chamber may also include additional passive and/or activefeatures to promote a facile and reproducible break of a swab within thesample chamber. Passive features may include one or more of, e.g.,geometrical configuration/arrangement of the sample chamber itself(e.g., curvature or angles along the length of the sample chamber),force focusing elements (e.g., protrusions from the internal walls ofthe sample chamber), and the like. Active features may include one ormore actuatable mechanisms arranged and configured within the samplechamber for cleaving the swab, e.g., a “guillotine” device similar to acigar cutter that can be actuated by a user exerting a force upon thedevice.

FIG. 29 shows sample chamber 2910, an adaptation of sample chamber 2810.Sample chamber 2910 has a constriction defined by protrusions 2990 thatproject inward from the walls of the chamber to form force focusingelements within the chamber. As illustrated in the figure, applying alateral force to swab 2930 that is seated in sample chamber 2910 causesthe swab shaft to contact one or more protrusions 2990. The lateralforce is thereby focused on one location on the swab, promoting breakageof the swab at that location. Preferably, the swab and sample chamberare designed/selected so that the swab has a weak point (shown as weakpoint 2937) at the same location (preferably, the swab is scored at thatlocation).

In an especially preferred embodiment, the sample chamber is configuredto cause an applicator stick to bend upon insertion thus promotingfracture of the shaft. FIG. 30 shows sample chamber 3010, an especiallypreferred adaptation of sample chamber 2810 that has a bend or angle3015 along its length such that the sample chamber has a first elongatedregion (on one side of the bend or angle) oriented in one direction anda second elongated region (on the other side of the bend or angle)oriented in second direction, the two regions being oriented at an anglerelative to each other. As shown in the FIG. 30, insertion of swab 3030leads to contact between a location on the shaft of the swab and a siteon the inner surface of the angle or bend. This contact focuses force onthat location and promotes breakage of the shaft at that location (toform head segment 3071 and shaft segment 3072). Preferably, the width ofthe sample chamber is designed to fit the swab head snugly but not sotightly that insertion of the swab requires excessive force. Mostpreferably, the swab and sample chamber are designed/selected so thatthe swab has a weak point (shown as weak point 3037) at or near thelocation of contact (preferably, the swab is scored at that location).Applicants have found that this arrangement allows for concurrentinsertion and breaking of the swab in one simple operation.Advantageously, the breakage is extremely reproducible and occurswithout any violent motion that can lead to expulsion of sample from thecartridge. Preferred angles or degrees of curvature are 20-90 degrees,more preferably 30-70 degrees, even more preferably 40-50 degrees, mostpreferably 45 degrees. While FIGS. 28, 29 and 30 illustrate embodimentsemploying swabs, the techniques are applicable to other types ofapplication sticks.

The reagent chambers are chambers adapted to hold liquid reagents usedduring the course of assays carried out in a cartridge. The reagentchamber design considerations for preferred embodiments of a cartridgedepend, in part, upon the particular assay(s) to be performed by thecartridge. For example, a cartridge may have one, two or more reagentchambers depending on the number of reagents required by the assayformat. Liquid reagents that may be held in a reagent chamber includebuffers, assay diluents, solutions containing binding reagents (e.g.,proteins, receptors, ligands, haptens, antibodies, antigens, nucleicacids and the like), solutions containing enzymes and/or enzymesubstrates, solutions containing control reagents, ECL read bufferscontaining ECL coreactants (e.g., tertiary amines such aspiperazine-N,N′-bis(2-ethanesulfonic acid) and tripropylamine), washsolutions, anti-foam agents, extraction reagents (e.g., solutionscontaining detergents, acids, bases, nitrous acid, nitrate salts, etc.)and the like. A cartridge may have one, two or more reagent chambersdepending, e.g., on the number of reagents required by the assay format.The reagent chamber design considerations for preferred embodiments of acartridge depend, in part, upon the particular assay(s) to be performedby the cartridge. The reagent chamber is connected to a reagent conduitfor transferring reagent from the chamber to other fluidic components inthe cartridge. The reagent chamber is, preferably, also connected to areagent vent port (optionally, through a reagent vent conduit). Thearrangement of the conduit connections to the chamber falls undersimilar design considerations as those described for the sample chamber,sample conduit and sample port; preferably, the reagent conduitintersects the chamber at or near the bottom and the reagent vent/ventconduit intersects the chamber at or near the top (relative to theorientation of the cartridge during use). Optionally, a filter elementis placed before or in the reagent conduit, e.g., if the reagentsolution is expected to contain particles that may clog the cartridgefluidics or otherwise negatively affect assay performance.

In one embodiment of the invention, a cartridge has one or more reagentcompartments that are empty or contain only dried reagents. Prior toconducting an assay, the user or cartridge reader dispenses liquidreagents into the these chambers (e.g., through reagent vent ports orthrough reagent introduction ports similar to the sample introductionport described above) which, optionally, reconstitute any dried reagentpresent in the chambers; the reagents are thus prepared for use in theassay. Sealable closures may be used to prevent leakage of the reagentsafter their addition.

Preferably, where an assay requires the use of liquid reagents, some orall of these liquid reagents are stored in liquid form in reagentchambers so as to minimize the number and complexity of the operationsthat must be carried out by a user or cartridge reader. In one preferredembodiment the reagent chamber(s) can be filled with the requisite assayreagent(s) at the time of cartridge manufacture and subsequently sealed.When used to store liquid reagents, the reagent chambers should bedesigned so as to prevent leakage and or evaporative loss of thereagents from the chambers during storage. In a particularly preferredembodiment the assay reagents are incorporated into assay reagentmodules that can be assembled into the cartridge's assay reagentchambers during manufacture. By designing the assay modules to havedesired properties such as resistance to leakage and evaporative loss,the design and manufacture of the rest of the cartridge is greatlysimplified. In such a preferred embodiment, an assay reagent releasemechanism would preferably be incorporated within the cartridge readerfor releasing the assay reagent from the reagent module. The assayreagent release mechanism is preferably adapted and configured to engagethe reagent module and release/recover its contents.

The reagent module is a container such as an ampoule (e.g., glass,plastic, or the like), a pouch (e.g., plastic, metal foil, plastic/metalfoil laminates, rubber, or the like), a blister pack, a syringe, or thelike, or any other container that can be filled with fluid, sealed anddropped into the cartridge for subsequent fluid delivery. Preferredmaterials include glass, plastics with good water vapor barrierproperties (e.g., cyclic olefin copolymers such as copolymers ofethylene and norbornene, nylon 6, polyethyelene naphthalate,polyvinylidene chloride and polychlorotrifluoroethylene) and metalfoil/plastic laminates because of their chemical inertness and theirresistance to evaporative losses, other suitable materials will beapparent to the skilled practitioner. Ampoules, preferably, comprise amaterial that can be made to shatter or break on impact such as glass orhard plastic. Embodiments incorporating breakable ampoules preferablyalso include filters to ensure that substantially all of the fragmentsthat may result upon rupturing the ampoules are not permitted to enterthe fluidic network and possibly obstruct/block fluid flow. FIG. 19depicts a cutaway top view of a cartridge showing filters 1515,1516 atthe bottom of chambers 1510 and 1511. These filters may be integrallymolded/machined, etched/etc. into the corresponding chambers.Alternatively, as illustrated in FIG. 20 depicting a bottom view of acartridge body, the filters 2020,2021 may be separate components thatare incorporated into the corresponding chambers during themanufacturing/assembly process; e.g., filter inserts that can beinserted/snapped into a receptacle within the chamber that is arrangedand configured to engagingly receive the filter insert.

The assay reagent release mechanism for releasing the contents of abreakable ampoule may be a simple mechanical device that is actuated toexert a force onto the ampoule; e.g., deliver a sharp blow to theampoule thereby rupturing it and releasing its contents into the assayreagent chamber. FIG. 21 depicts one preferred embodiment of a reagentchamber employing assay reagent ampoules 2120,2121. Preferably, a coverlayer (not shown), most preferably made from a flexible material, issealed to the top of the cartridge body so that liquid does not leakfrom the cartridge after the ampoules are ruptured (see, e.g., coverlayer 1401 in FIG. 14). FIG. 21 also shows assay release mechanism 2110(preferably, a component of a cartridge reader) which can be actuated sothat hammer element 2115 strikes an ampoule, preferably by striking aflexible cover layer that then transfers the impact force to the ampoule(while, preferably, remaining intact so that it confines the releasedliquid to the reagent chamber). It has been observed that striking theampoule quickly with an adequate impulsive force produces a morecomplete rupturing of the ampoule and thereby more effectively releasingthe assay reagent. Whereas a slowly applied force increasing inmagnitude until ultimately the ampoule fractures results in lesscomplete rupture and less effective assay reagent release.

In an alternative embodiment, a pierceable container such as a pouch orblister pack may be employed. Preferably, the pierceable container has apierceable wall made from a plastic film, a metal foil, or mostpreferably, a metal foil/plastic film laminate. In such an embodimentthe assay reagent release mechanism could employ a piercing scheme. FIG.22 shows an exploded view of one preferred embodiment of a reagentchamber for holding a pierceable container. Reagent chamber 2210 haspiercing tip 2212 located at the bottom of the chamber. Chamber 2210 isconnected to reagent conduit 2216 and, optionally, a vent conduit (notshown). Reagent module 2220 comprises module body 2230, preferably madeof injected molded plastic, that defines the walls of a fluidcompartment, having a first opening 2232 and a second opening 2234.Fluid is sealed in the compartment by first opening cover 2242 andsecond opening cover 2244, the covers preferably made of a plastic-metallaminate (most preferably and aluminum coated mylar film). Module 2220also, preferably, has tongue 2250 that fits in chamber groove 2214 so asto properly align module 2220 in chamber 2210 and hold module in anelevated position above piercing element 2212. Chamber 2210 also,preferably, has a chamber cover layer that prevents leakage of reagentfrom the chamber after rupture of module 2220. On application of athreshold downward force to module 2220, preferably through a flexiblechamber cover layer, module 2220 is pushed against tip 2212, piercingfirst opening cover 2242 and releasing the reagent into the chamber.Module 2220 also, preferably, comprises a second piercing tip 2236 thatis attached to the module walls via a cantilever (the second piercingelement and cantilever are preferably integral to the module body; sucha component is readily manufacturable, e.g., by injection molding). Whenpiercing tip 2212 pierces first opening cover 2242 in a module with asecond tip element 2236, piercing tip 2212 pushes second piercing tip2236 until it pierces second opening cover 2234 making a second openingin module 2220 and facilitating extraction of the fluid from the pouch;i.e., venting the pouch itself.

In another alternate embodiment, liquid reagents are stored in a syringecomprising a syringe chamber and a plunger. The chamber may be anintegral component of the cartridge, a module that is inserted into thecartridge or a separate component that is attached (e.g., via a luerlock connection) to the cartridge prior to use. Actuation of the plungermay be used to release the contents of the syringe into a reagentchamber or, alternately, to transfer the contents directly into otherfluidic components of the cartridge.

An important consideration for cartridge based assay systems relates tolong term storage of the cartridge prior to use; i.e., “shelf life” ofthe cartridge. Certain assay reagents (especially biological reagentsand/or binding reagents such as enzymes, enzyme substrates, antibodies,proteins, receptors, ligands, haptens, antigens, nucleic acids and thelike), when dissolved in a liquid medium require special handling andstorage in order to improve their shelf life. In certain instances, evenif the assay reagents dissolved in liquid media are handled and storedin strict compliance with the special handling and storage requirementstheir shelf life is impracticably short. Furthermore, the need toobserve special handling and storage requirements adds to the complexityand cost of the cartridge based system employing such reagents. Thespecial handling and storage requirements can be substantially reduced,if not eliminated, and the complexity and cost of the system can beminimized by using more stable dry, or dehydrated, forms of the assayreagents. The use of dry reagents can also simplify mixing operationsand reduce the volume and weight of a cartridge. Reagents that may beincluded in dry form include biological reagents, binding reagents, pHbuffers, detergents, anti-foam agents, extraction reagents, blockingagents, and the like. The dry reagent may also include excipients usedto stabilize the dry reagents such as sugars (e.g., sucrose ortrehalose). For assays may encounter acidic or basic samples (e.g.,samples that are inherently acidic/basic and/or samples that areextracted or otherwise treated with an acidic/basic reagent), a dryreagent may include a neutralizing reagent (e.g., an acid, base of a pHbuffer). In especially preferred embodiment that involve extraction ofsamples with nitrous acid, the extracted sample is passed over a dryreagent comprising a base or, more preferably, the base form of abuffering agent (e.g., Tris, Hepes, phosphate, PIPES, etc.). Asufficient amount of the base or buffering agent is included to bringthe pH of the extracted sample to a value that is compatible withsubsequent assay reactions carried out on the sample (e.g., bindingreactions with binding reagents).

Dry reagents may be employed in a cartridge based assay system in anumber of ways. As described above, dry reagents may be stored in areagent chamber that is filled prior to use by a user or by a cartridgereader apparatus. Similarly, dry reagents may be stored in other fluidiccomponents such as within fluidic conduits or chambers, most preferablywithin a fluidic conduit connecting the sample and detection chambers.Introduction or passage of liquid (e.g., a liquid sample or a liquidreagent) through the conduit or chamber results in dissolution of thedry reagent. Dry reagents may be inserted during the manufacture of acartridge by depositing the dry reagents in the appropriate fluidiccomponent, e.g., by depositing the reagent in the form of a powder orpellet or by incorporating the dry reagent in a screen printed ink.Alternatively, the reagents may be inserted in solution and then driedto remove the solvent. In one preferred embodiment dried reagents may beformed upon a substrate by depositing solutions containing the reagentsin one or more predefined locations and subsequently drying the reagentsto form a dried reagent pill under conditions such that on addition of aliquid sample or an appropriate solvent, the dry reagent dissolves intosolution. The term “pill” is used herein to refer generally to an amountof a dry, but redissolvable, reagent on a substrate and not to connoteany specific three dimensional shape. The location of a pill on asubstrate is referred to herein as a “pill zone”. The substrate ispreferably a component of the cartridge, e.g., cartridge body, chamber,cover layer, electrode array, etc. Suitable locations for the pill zoneinclude the sample chamber, reagent chamber, sample conduits, andreagent conduits so that liquid reagents and samples pick up the dryreagent prior to their introduction to the detection chambers.Alternatively, the reagent pills may be located within the detectionchambers themselves. In the preferred embodiment depicted in FIG. 13 a,the dried reagent pills are formed upon the cover layer 1322 in twopredefined pill zones. In another preferred embodiment, a reagentchamber holds a liquid reagent in an ampoule and a dry reagent pill, sothat the dry reagent is reconstituted upon rupture of the ampoule. Thisarrangement is useful for preparing a reagent containing a reactivecomponent. In one example, the ampoule contains an acid such as aceticacid and the dry reagent is a nitrate salt so that rupture of theampoule results in the preparation of nitrous acid.

A pill zone in which dried reagents are deposited may be prescribed by aboundary which confines the volume of a deposited solution (and,therefore, the dried reagent left after allowing the solution to dry) toa specific region of a substrate. According to one preferred embodimentof the invention, a cartridge comprises a pill zone that is bounded by aboundary surface, the boundary surface being raised or lowered(preferably, raised) and/or of different hydrophobicity (preferably,more hydrophobic) than the pill zone. Preferably, the boundary surfaceis higher, relative to the substrate surface within the pill zone, by0.5-200 micrometers, or more preferably by 2-30 micrometers, or mostpreferably by 8-12 micrometers. Even more preferably, the boundarysurface has a sharply defined edge (i.e., providing a steep boundarywall and/or a sharp angle at the interface between the pill zone and theboundary). Preferably, the pill zone surface has a contact angle forwater 10 degrees less than the boundary surface, preferably 15 degreesless, more preferably 20 degrees less, more preferably 30 degrees less,even more preferably 40 degrees less, and most preferred 50 degreesless.

In one preferred embodiment the pill zone is defined by a depression cutor molded into the substrate. In another embodiment, the boundarysurface around a pill zone is defined by a boundary material applied onthe substrate. In one example, the pill zone is defined by a cutout in afilm or gasket applied to the substrate, preferably a cutout in a filmof adhesive tape. In another preferred embodiment the boundary can bephysically defined by applying a coating in a manner which defines theboundary of the pill zone using, e.g., established techniques forforming patterned coatings such as photolithography, patterneddeposition, screen printing, etc. In one example, a patterned dielectriccoating can be screen-printed onto the surface of a substrate material,the pattern including apertures, the boundaries of which define the pillzone. The reagent can then be dispensed onto the substrate within thepill zone boundary and thereafter dried to form the dried reagent pill.

The waste chambers are chambers adapted to hold excess or waste liquid.In certain embodiments, the detection chamber may also act as a wastechamber. In certain embodiments, however, it is beneficial to have aseparate waste chamber, e.g., when carrying out assay formats thatinvolve passing samples through the detection chamber having a volumegreater than the volume of the detection chamber or when carrying outassay formats that involve wash steps to remove sample from thedetection chamber. Sizing of the waste chambers is preferably done inaccordance to the anticipated volumes of sample and liquid reagents thatwill be used in the assay. Another sizing related factor for the wastechambers that is preferably taken into account relates to the potentialfor waste fluids, as they enter the waste chamber to foam or bubble. Insuch instances, where foaming or bubbling is anticipated, the wastechamber volume could be increased sufficiently to avoid any issues thatcan arise from such foaming or bubbling.

Waste chambers are linked to a waste chamber conduit and, preferably, toa vent port (e.g., through a vent conduit). The waste chamber isconfigured to allow liquid waste to be delivered to the waste chamberthrough the waste chamber conduit and, preferably, for air that isincluded in the waste stream to escape through a waste chamber ventport. Optionally, the waste chambers contain a water absorbing material,such as a sponge, that retains waste fluid and prevents leakage of thewaste fluid on disposal of a cartridge. A factor that is preferablyconsidered when designing the configuration and arrangement of the wastechambers relates to eliminating or substantially reducing thepossibility that fluid from the waste chamber can flow back(“back-flow”) into the cartridge's fluidic network. In particularlypreferred embodiments, as illustrated in FIG. 10, the waste chamberconduits are arranged/routed such that they are fluidically connected tothe waste chambers at points 1040,1041 that are above the anticipatedfill levels/lines (i.e., the fill level/line is defined by the volume ofwaste fluid that resides within the waste chamber at the conclusion ofthe assay). This preferred configuration substantially reduces oreliminates the possibility that fluid from the waste chamber can flowback (“back-flow”) into the cartridge's fluid network.

The issue of back-flow may also arise in the context of bubbling/foamingof the waste fluids. The vent port is preferably linked via a conduitwith a large enough volume to allow a small amount of liquid to enterthe conduit (e.g., because of foam in the waste chamber) without thisliquid reaching the vent port (as described for above for the samplechamber). Furthermore, aerosol-prevention plugs or gas-selectivemembranes (i.e., materials that selectively allow the passage of gas butprevent the passage of liquids) may be included into the waste chambervent conduits or vent ports to prevent release of liquid through thesepassages. Aerosol-prevention plugs are commonly used in pipette tips toprevent contamination of pipettors and include materials that allow thepassage of air when dry but swell and seal up the passage when they comein contact with liquid (e.g., filter materials impregnated or coatedwith cellulose gum).

An additional measure for eliminating or substantially reducingfoaming/bubbling of waste fluids as they are introduced into the wastechamber can be employed in particularly preferred embodiments. Such anadditional anti-foaming/bubbling measure may include arranging/routingthe waste chamber conduit such that it enters the waste chamber at aposition that is located above the fill line and that intersects avertical wall of the waste chamber, as illustrated by conduit segments910 and 911 entering waste chambers 930 and 931 in the embodimentdepicted in FIGS. 9 and 10. Such a configuration allows the waste fluidto be introduced into the waste chamber in a manner so as to allow thefluid to run along a vertical wall of the waste chamber. Advantageously,this substantially reduces or eliminates foaming/bubbling of the wastefluid as it is routed into the waste chamber.

Yet another anti-foaming/bubbling measure that may be employed incertain preferred embodiments comprises a vertical web, or partial wall,that can be included in the upper portion of the waste chamber. Aparticularly suitable embodiment for inclusion of such ananti-foaming/bubbling measure is the two-piece cartridge body designdepicted in FIG. 16. The anti-foaming web/wall is preferably included inthe upper portions of the waste chambers 1610,1611 located in the uppercartridge component 1500. Preferably the anti-foaming web is arrangedbetween the waste chamber vent and the waste chamber input. The heightof the anti-foaming web preferably extends the fill depth of the upperportion of the waste chamber but may be less than the full depth aswell. Alternatively, the anti-foaming web can extend beyond the depth ofthe upper portion of the waste chamber so that it protrudes into thelower portion of the waste chamber. Preferably the height of theanti-foaming web is selected to achieve optimum anti-foaming by allowingthe flow of liquid under the web/wall but blocking the flow of bubblesabove the surface of the liquid in the waste chamber.

Yet another anti-foaming/bubbling measure is to include an anti-foamagent in the waste chamber or in another conduit or chamber of thecartridge so that liquid entering the waste chamber has less propensityto foam and/or form bubbles.

The detection chambers are adapted for carrying out a physicalmeasurement on the sample. The detection chamber is connected to aninlet conduit. Preferably, the detection chamber is also connected to anoutlet conduit and is arranged as a flow cell. If the measurementrequires illumination or optical observation of the sample (e.g., as inmeasurements of light absorbance, photoluminescence, reflectance,chemiluminescence, electrochemiluminescence, light scattering and thelike) the detection chamber should have at least one transparent wallarranged so as to allow the illumination and/or observation. Whenemployed in solid phase binding assays, the detection chamber preferablycomprises a surface (preferably, a wall of the chamber) that has one ormore binding reagents (e.g., antibodies, proteins, receptors, ligands,haptens, nucleic acids, etc.) immobilized thereon (preferably, an arrayof immobilized binding reagents, most preferably an array of immobilizedantibodies and/or nucleic acids). In an especially preferred embodiment,the detection chamber is an electrochemiluminescence detection chamberas described above, most preferably having one or binding reagentsimmobilized on one or more electrodes. In one preferred embodiment, thecartridge comprises a working electrode having an array of bindingreagents immobilized thereon. In another preferred embodiment, thecartridge comprises an array of independently controllable workingelectrodes each having a binding reagent immobilized thereon.Preferably, in cartridges employing arrays of binding reagents, at leasttwo elements of the array comprise binding reagents that differ inspecificity for analytes of interest. Suitable detection chambers,electrode arrays and arrays of immobilized binding reagents for use inECL-based cartridge systems are described in detail above and includethe embodiments shown in FIGS. 1-4.

The detection chamber is, preferably, arranged in an elongated flow celldesign with inlet and outlets at or near opposing ends of the elongateddimension. Depending on the application, manufacturing approach, samplesize, etc., the flow cell dimensions can range from nanometers to tensof centimeters and the volume from picoliters to milliliters. Certainpreferred embodiment have widths that can range from 0.05-20 mm, morepreferably, 1-5 mm and heights (preferably, less than or equal to thewidth so as to increase, for a given volume, the surface area of thebottom of the detection chamber, especially when this surface is used toimmobilize binding reagents) that range from 0.01-20 mm, morepreferably, 0.05-0.2 mm. Preferably, the height is less than or equal tothe width. Preferably, the detection chamber is designed to accommodatesample volumes between 0.1-1000 uL, more preferably, 1-200 uL, morepreferably, 2-50 uL, most preferably, 5-25 uL. In embodiments that arelimited by sample volume (e.g., cartridges measuring blood from fingerpricks), especially preferred detection chamber volumes are less than 10uL, more preferably 0.5-10 uL, even more preferably 2-6 uL. The flowcell preferably has a width greater than or equal to the height.

A cartridge may comprise one or more detection chambers. Cartridgescomprising multiple detection chambers may comprise separate fluidicsystems for each detection chamber (e.g., multiple sample chambersand/or reagent chambers and associated fluidic conduits) so that assayson multiple samples may be carried out in parallel. In certain preferredembodiments, multiple detection chambers are linked to a single samplechamber and may share the use of other fluidic components such asreagent chambers, waste chambers and the like. In these embodiments, thetwo detection chambers may be used to carry out different sets ofassays, thus increasing the number of measurements that can be carriedout on a sample relative to a cartridge with one detection chamber.Advantageously, the use of multiple detection chambers allows forcarrying out in a single cartridge multiple incompatible measurements,that is measurements that can not be performed in a single reactionvolume or benefit from being carried out in separate reaction volumes,e.g., measurements that have different requirements for pH or assaycomposition or otherwise negatively interfere with each other.

In an alternate embodiment employing a plurality of detection chambers,one or more of a plurality of detection chambers is used ascontrol/calibration chamber for measuring assay control/calibrationsamples. In one such embodiment, a first and a second detection chamberare each configured to carry out a panel of one or more assays for oneor more analytes. One detection chamber (the test chamber) is used toanalyze a sample. The other detection chamber (the control chamber) isused to analyze a spiked sample having a predetermined additional amountof the one or more of the analytes of interest (this predeterminedadditional amount, preferably, being provided by passing the samplethrough a reagent pill zone comprising the additional amounts). Thechange in signal between the two chambers allows for the calculation ofthe responsivity of the signal to changes in analyte and can be used tocalibrate the system and/or to determine if the cartridge is functioningproperly. In another embodiment employing a control chamber, the controlchamber is not used to analyze the sample or a derivative thereof but isused to measure analyte in a separate control or calibrator matrix. Thesignal in the control chamber may be used for determining backgroundsignals (by using a matrix with no analyte), for calibrating theinstrument (by using a calibrator matrix with a predetermined amount ofanalyte to determine calibration parameters) or to determine if thecartridge is functioning properly (by using a control matrix with apredetermined amount of analyte and determining if the signal fallswithin a predetermined acceptable range).

The cartridge fluidics may include bubble traps. The bubble trap is achamber or conduit adapted for removing bubbles from fluid streams.Preferably, there is a bubble trap between the sample and detectionchambers so that bubbles in the sample may be removed prior tointroducing the sample into the detection chamber. FIG. 31 shows across-sectional view of one exemplary embodiment and shows bubble trapchamber 3110 connected to inlet conduit 3140 and outlet conduit 3145(the inlet and outlet conduits being, preferably, located near thebottom of chamber 3110) and vent port 3150. Liquid is introduced intochamber 3110 via inlet 3140. Chamber 3110 is, preferably, wide enough sothat bubbles in a liquid introduced to the chamber can rise to the topof the chamber and be expelled via vent port 3150. Bubble-free liquid isthen expelled via outlet 3145. Optionally, outlet conduit 3145 isomitted; in this case a liquid is admitted via inlet conduit 3140,bubbles are expelled via vent port 3150 and the liquid is then expelledback through inlet conduit 3140. Optionally, an air-permeable butwater-impermeable membrane (e.g., a membrane made from Gortex material)is placed between inlet 3140 and vent port 3150. When a liquid passesthrough the conduit that contains bubbles or is present in a stream thatis segmented by slugs of gas, the gas/bubbles will pass through themembrane and exit through vent port 3150 (preferably, the process isaided by applying suction at vent port 3150) to ensure that liquid isnot expelled via vent port 3150 (the optional membrane is shown asmembrane 3190).

The fluidic conduits can be located at any position within the cartridgeand oriented at any angle. Advantageously, the fluidic channels arelocated, primarily, in planar networks, preferably located proximate tothe outside surfaces (e.g., the top 901,902 or bottom 903 surfaces ofthe cartridge shown in FIGS. 11 a-c) to allow for a multi-layeredcartridge design that uses, e.g., machined, die-cut, laser-cut and/ormolded cartridge body components. Preferred conduit geometries includeconduits with cross-sections that are circular, oval, square orrectangular in cross-section. The width is, preferably, similar to theheight so as to minimize the surface area for a particularcross-sectional area. Width and height can vary widely from nm to cmranges depending on the application, sample volume and cartridge design.Preferred ranges for the width and height are 0.05 to 10 mm, morepreferably, 0.5 to 3 mm, most preferably 1 to 2 mm. Cartridges adaptedto low volume samples such as blood from finger pricks may have smallconduits, preferably having height/widths <1 mm, preferably between 0.4to 1.0 mm.

The fluidic channels preferably make use of “z-transitions” that routethe fluid flow path between planes. A conduit with such a z-transitionmay comprise first, second, and third conduit segments arranged insequence, the first and third conduit segments being located indifferent planar fluidic networks and the second conduit segmentconnecting the two fluidic networks and arranged at an angle to theother two segments. By way of example, “z-transitions” (denoted in FIG.9 as capillary breaks) route the fluid flow/path, in the cartridge shownin FIGS. 11 a-c, from fluidic conduits near the upper surface 901,902 tofluid conduits near the bottom 903 surface and vice a versa.Z-transitions are advantageous in that they provide capillary breaks (asdescribed below) and allow for more complicated fluidic networks thanwould be possible if the fluidic conduits were confined to one plane.Selective use/placement of capillary breaks, preferably z-transitions,may be used to control the passive flow of fluids and prevent mixing offluid streams. Certain preferred embodiments of the invention employ“double z-transitions”, that is conduits that comprise a firstz-transition that directs fluid flow from a first planar network to asecond planar network, a second z-transition that redirects fluid flowback to the first planar network and a connecting segment in the secondplanar network that connects the two z-transitions. Such a doublez-transition may comprise first, second, third, fourth and fifth conduitsegments arranged in series, the first and fifth segments located in afirst planar fluidic network, the third segment located in a secondplanar fluidic network, the second and fourth segments located so as todirect flow between the two planar networks.

The fluidic network may be formed within the cartridge in a number ofdifferent ways, dependent, in part, upon the materials chosen for thecartridge. Any known fabrication method appropriate to the cartridgebody material may be employed including, but not limited to,stereolithography, chemical/laser etching, integral molding, machining,lamination, etc. Such fabrication methods may be used alone or incombination. In certain embodiments of the invention, the cartridgecomprises a cartridge body and one or more cover layers mated tosurfaces of the cartridge body so as to define one or more fluidicnetworks preferably, planar fluidic networks) therebetween. Similarly,z-transitions and/or ports can be selectively molded into, or machinedout of, the cartridge body at predetermined locations to form thefluidic connections between the channels on the upper and lowersurfaces.

One preferred embodiment of the cartridge may be fabricated using a“lamination” process whereby the cartridge body's functional surfacesare sealed using cover layers to form the fluidic network. For example,recesses (e.g., channels, grooves, wells, etc.) one or more surfaces ofthe cartridge body to provide what is referred to herein as “functionalsurfaces”. Sealing/mating of the functional surfaces to cover layersforms a fluidic network comprising fluidic components (e.g., conduits,chambers, etc.) at least some of which are defined in part by therecesses in the cartridge body and in part by a surface of a coverlayer. The cover layers are preferably comprised of plastic film such asmylar film. The cover layer may be coated with an adhesive to seal thecover layer against the cartridge layer. Other methods for mating thecover layer to the cartridge body will be known to the skilled artisan,e.g., the seal may be achieved by heat sealing, ultrasonic welding, RF(radio frequency) welding, by solvent welding (applying a solventbetween the components that softens or partially dissolves one or bothsurfaces), by use of an intervening adhesive layer (e.g., a double sidedadhesive tape, etc.). Advantageously, cartridge features that arecreated by patterned deposition (e.g., patterned deposition of electrodeor dielectric layers and/or patterned deposition of reagents to form dryreagent pills or to form binding domains with immobilized bindingreagents) are created on cover layers so as to take advantage ofautomation available to process plastic film in large sheets or rolls.

Recesses may be, e.g., molded in, etched in or machined from thecartridge body. By analogy, fluidic components may also be defined, atleast in part, by recesses in a cover layer that is mated to a cartridgebody. Fluidic components may also be defined, at least in part, byregions cutout from gasket layers disposed between the cartridge bodyand cover layers. Apertures in the cartridge body and/or cover layersmay be used to provide for access ports to the fluidic network, e.g.,sample introduction ports, vent ports, reagent addition ports and thelike. Vent ports, preferably, allow the equilibration of fluid in thechambers with the atmosphere or to allow for the directed movement offluid into or out of a specified chamber by the application of positiveor negative pressure. Vent ports, preferably, are designed to preventthe leakage of liquid samples or reagents through the ports and mayinclude aerosol-resistance filters, membrane or filter materials thatpermit air flow but act as barriers to aqueous solutions (e.g., filteror membranes made from porous hydrophobic materials such as Gortex), andmaterials that are porous to air but seal when they come in contact withaqueous solutions (e.g., cellulose gum impregnated filters).

Preferred embodiments include a cartridge having a cartridge body with afirst side and a second, preferably opposing, side and one or more coverlayers mated to the first side to form a first fluidic networktherebetween and one or more cover layers mated to the second side toform a second fluidic network therebetween. Through-holes through thecartridge body (which may be formed by molding, etching, machining,etc.) may be used to link the first and second fluidic networks and toprovide Z-transitions. Additional fluidic complexity can be built into acartridge by employing a laminated cartridge body having multiplecartridge body layers and additional fluidic networks between theselayers; through-holes through the various cartridge body layers are usedto link the different fluidic networks.

A high degree of control over the movement of liquids in the cartridgesof the invention may be attained, without the introduction of activevalve elements in the cartridge, through the use of fluidic networkscomprising capillary breaks. “Capillary break”, as used herein, refersto a region in a fluid conduit that acts as a barrier to liquid movingthrough the conduit under capillary action or under the driving force ofa low pressure gradient below a threshold pressure. In preferredexamples of capillary breaks, application of a pressure above thethreshold pressure acts to push the fluid past the barrier. Capillarybreaks may be designed into fluid conduits by introducing, e.g., i) atransition, on a surface of a conduit, from a wettable surface to a lesswettable surface (e.g., as indicated by the contact angle for water);ii) a transition in conduit width from a region of narrow width thatpromotes capillary flow to a region of wider width; iii) a transition,on a surface of a conduit, in roughness; iv) a sharp angle or change indirection and/or v) a change in cross-sectional geometry. In anotherembodiment, a fluid conduit has a flexible wall/diaphragm that impingesinto the conduit and blocks flow driven by a pressure below a thresholdpressure. Application of a higher pressure forces the flexiblewall/diaphragm out of the flow path and lets fluid flow. Preferably, thediaphragm is made of a material (e.g., Gortex) that allows gas to passthrough but prevents the flow of liquid up to a certain pressure.Preferred capillary breaks involve a sharp angle or change in directionin a fluid conduit, most preferably a “Z-transition” as described above.

In one embodiment of the invention, a liquid is introduced into achamber comprising an outlet conduit that includes a capillary break(preferably a Z-transition). The liquid enters the outlet conduit butstops at the z-transition. A pressure gradient is then applied (e.g., byapplying positive pressure to the chamber or negative pressure to theother end of the conduit) which cause the liquid to flow past thez-transition into the rest of the conduit.

The fluidic network may also comprise valves to control the flow offluid through the cartridge. A variety of suitable valves (includingmechanical valves, valves based on electrokinetic flow, valves based ondifferential heating, etc.) will be known to one of average skill in theart of assay cartridges or microfluidic devices. In preferredembodiments, however, at least one and more preferably all activelycontrolled valve elements are external to the cartridge. In oneembodiment, a fluid conduit has a flexible wall/diaphragm that in theabsence of external force allows fluid to pass through the conduit.Application of an external force on the wall/diaphragm (e.g., from apiston or via the application of gas or hydrostatic pressure) causes thediaphragm to impinge on the conduit, thus impeding the flow of fluid.

The fluidic network may include at least one viscosity measuringconduit, preferably linked to a sample chamber or sample conduit, havingan inlet and an outlet. The conduit is adapted so that a liquid samplecan be introduced into the conduit and the time it takes the liquid tomove between two locations in the conduit can be timed (preferably usingsensors such as impedance sensors or optical sensors in the cartridge oran associated cartridge reader). Such an arrangement can advantageouslybe used to measure clotting times of a blood or plasma sample. Formeasuring clotting times, the conduit or an upstream componentpreferably comprises a dry reagent necessary for the specific clottingmeasurement (e.g., activated clotting time, whole blood clotting time,prothrombin time, thrombin time partial thromboplastin time and thelike).

Vent ports as described above are, preferably, apertures on the surfaceof the cartridge that are in fluidic communication with fluidic chambersor conduits within the cartridge. In a laminated cartridge construction,the vent ports may be provided, for example, by apertures in coverlayers that seal against a cartridge body to define planar fluidicnetworks or alternatively, by through-holes exposed on one surface ofthe cartridge body that communicate with fluidic networks on theopposing side. The vent ports act as control ports that allow acartridge reader to control the movement of fluid in the cartridge,e.g., by a combination of sealing one or more ports, opening one or moreports to atmospheric pressure, connecting one or more ports to a sourceof positive pressure and/or connecting one or more ports to a source ofnegative pressure. The vent ports may also be used to introduce air intoliquid streams passing through the fluidic conduits of the invention,for example, to segment the fluid streams with slugs of air. Theintroduction of air may be used to prevent mixing of two liquid slugspassed sequentially through a conduit, to clear a liquid from a conduitand/or to enhance the efficiency of a wash step. Preferably, the ventports are arranged in a single row at a common location along thecartridge body's width. Such an arrangement and configuration of thecontrol points advantageously allows the interface between the cartridgereader and the cartridge to be simplified. For example, using such apreferred configuration allows the cartridge reader to make use of asingle fluidic mating device for placing the cartridge into fluidiccommunication with the cartridge reader. Such a configuration alsoallows the motion control subsystem(s) to be simplified in that a singlemotor or actuation device may be used to actuate the fluidic matingdevice and move it into scaling engagement with the cartridge body.

FIG. 9 is a schematic representation of cartridge 900, one preferredembodiment of a cartridge of the invention that incorporates many of thefluidic features described above. This exemplary embodiment depicts acartridge comprising an electrode array of the invention as describedabove. The skilled artisan, however, can readily adapt the fluidiccomponents and design to cartridges employing other detection chamberdesigns and/or detection technologies. The cartridge schematic shown inFIG. 9 comprises various compartments including a sample chamber 920,assay reagent chamber 925, waste chambers 930 and 931 and detectionchambers 945 and 946 comprising electrode arrays 949 a and 949 b andelectrode contacts 997 and 998. Also depicted in FIG. 9 are fluidports/vents 950-953 and 980 that may be utilized as fluidic controlpoints, vents for allowing a chamber to equilibrate with atmosphericpressure, ports for introducing air bubbles or slugs into a fluid streamand/or as fluidic connections to a cartridge reader. FIG. 9 also depictsa number of fluidic conduits (shown as lines connecting the variouschambers) that establish a fluidic network that connects the variouscompartments and/or fluid ports/vents. The fluidic conduits may comprisedistribution points (e.g., branch points such as distribution point 976that are adapted to distribute a fluid to two or morelocations/compartments in a cartridge). Other fluidic features that areshown in FIG. 9 include pill chambers/zones 990,991 for each of the readchambers. FIG. 10 depicts a three dimensional representation of thefluidic network formed by the various fluidic components employed in apreferred embodiment of FIG. 9.

Sample chamber 920 is a chamber defined within cartridge 900 that isadapted for receiving a sample, preferably a liquid sample, to beanalyzed in the cartridge. Sample chamber 920 includes a sampleintroduction port 921, and is linked to vent port 953 through a ventconduit and detection chambers 945 and 946 through sample conduit 901having sample conduit branches 940 and 941. Preferably, cartridge 900also includes a sealable closure for sealing sample introduction port921. Reagent chamber 925 is a chamber adapted to hold a liquid reagentand includes a vent conduit linked to vent port 950 and reagent conduit902 linked to the sample conduit (preferably, between sample chamber 920and distribution point 976). Also linked to the sample conduit is airchamber/trap 975 linked to vent port 980. This arrangement allows foradding/removing air into/from the fluid stream(s) (e.g., to reagent orsample streams directed from reagent chamber 925 or sample chamber 920towards detection chambers 945 or 946) in the fluidic pathway byapplying positive pressure or suction to vent port 980. Pillchambers/zones 990 and 991 hold dry reagents and are positioned,respectively, in the fludic pathway between sample port 920 anddetection chambers 945 and 946 so that liquid passing through thechamber/zones will reconstitute the dried reagents and carry theresulting solutions into the detection chambers. Reagent chamber 925,air chamber trap 975, vent port 980 and/or pill chamber zones 990 and/or991 may optionally be omitted.

Detection chambers 945 and 946 are adapted for carrying out a physicalmeasurement on a sample, preferably an electrochemiluminescencemeasurement, most preferably a measurement employing an electrode arraythat is configured to be fired in a pair-wise fashion (as describedabove). Optionally, detection chamber 946 is omitted. As depicted in thepreferred embodiment of FIG. 9, detection chambers 945 and 946 havedifferent geometrical cross-sections than their respective input andoutput channels to which they are in fluidic communication. As such, itis preferable to incorporate transitional fluidic segments (947 a,b and948 a,b) at the inputs and outputs of the read chambers such that fluidflow may be appropriately transitioned between the dissimilar regions.Preferably, the transition is designed to minimize the transitionlength; e.g., incorporating a diffusers/nozzles with as wide an angle aspossible, while being gradual enough to prevent trapping of air bubbles.Detection chambers 945 and 946 are connected via waste conduits 960,961to waste chambers 931 and 930. Waste chambers 930 and 931 are chambersconfigured to hold excess or waste fluids and are also connected,respectively, to vent port 952 via a vent conduit and vent port 951 viaa vent conduit. The use of multiple waste chambers advantageously allowsfluid flow through the multiple chambers to be controlled independentlyvia the application of vacuum or pressure to the waste chamber ventports. Alternatively, only one waste chamber is used (e.g., wastechamber 930 is omitted and detection chambers 945 and 946 are bothconnected to waste chamber 931).

In cartridges for conducting binding assays for analytes of interest,pill zones 990 and 991 preferably comprise labeled binding reagents(e.g., antibodies, nucleic acids, labeled analogs of analytes ofinterest, etc.), detection chambers 945 and/or 946 comprise one or moreimmobilized binding reagents (preferably, an array of immobilizedbinding reagents, most preferably immobilized on electrodes forconducting ECL assays) and reagent chamber 925 comprises a wash reagentfor removing sample solution and/or unbound labeled reagents from thedetection chambers. In embodiments where one of the detection chambersis used for control assays or for assay calibration, the associated pillzone may comprise control reagents such as an added analyte (forexample, to be used in spike recovery, calibration measurements orcontrol assay measurements).

The fluidic network of cartridge 900 comprises z-transitions that mayact as capillary breaks and/or allow for the fluidic network to beextended to multiple planes of the cartridge. See, e.g., Z-transitions1010-1014 in FIG. 10. Z-transition 1011 in the sample conduit and 1013in the reagent conduit act as capillary breaks which confine sampleliquids and reagent liquids to their corresponding chambers. Fluid canbe moved from these chambers, in a controlled and reproducible manner,by application of an appropriate pressure gradient. Z-transitions 1060and 1061 allows the waste conduits to cross sample conduit branches 940and 941 by arranging them on different layers of the cartridge.

FIGS. 13 a and 13 b show exploded views of one embodiment of cartridge900 that comprises cartridge body 1100 and cover layers 1324, 1350,1320, 1321 and 1322 mated to the surfaces of cartridge body 1100. FIG.11 shows top (FIG. 11 a), bottom (FIG. 11 b) and isometric (FIG. 11 c)views of cartridge body 1100. The upper 1101,1102 and lower 1103surfaces of the cartridge body 1100 incorporate (e.g., by molding,machining, etching, etc.) recessed features such as channels, grooves,wells, etc. The features are sealed to provide the chambers and conduitsof the cartridge by applying the cover layers to the upper and lowerportions of the cartridge body. To allow for adequate sample and/orreagent volumes, the cartridge body has thicker portion 902 whichincludes features (channels, grooves, wells, compartments, etc.) thatdefine, in part, the sample, reagent and waste chambers. The remainderof the cartridge is, preferably, much thinner so as to minimizecartridge weight, volume and material costs and, in the case, of certainpreferred cartridge designs, to allow optical detectors to as close aspossible to the top surface of electrodes incorporated on a cover layeron the bottom of a cartridge.

Reagent chamber 925, sample chamber 920, waste chambers 930 and 931 andat least portions of the sample conduit, reagent conduit and wasteconduits 960 and 961 are formed by sealing cover 1324 on cartridge body1100. Detection chambers 945 and 946 are formed by sealing cover layer1350 (having patterned conductive layer 1360 (which forms the patternedelectrode array 963, shown in FIG. 9) and patterned dielectric overlayer1365) to cartridge body 1100 through intervening gasket layer 1331(preferably, made from double sided adhesive tape). The detectionchamber's depth, length and width are defined by cutouts 1340 and 1341within the gasket layer. Cover layer 1322 mates to cartridge body 1100through gasket layer 1330 (preferably a double sided adhesive tape) todefine conduit segments, such as 1060 shown in FIG. 10, that (viaformation of double z-transitions) act as bridge segments connecting thefluidic networks defined by cover layers 1324 and 1350. Advantageously,the use of a such a “bridge” cover layer allows cover layer 1350 havingpatterned electrodes (and, optionally, patterned binding reagents on theelectrodes) to be only slightly larger than the patterned components.This arrangement decreases the cost of the patterned component.Alternatively, the bridge cover layer and associated doublez-transitions can be omitted and cover layers 1324 and 1350 can becombined into a single contiguous cover layer. Optionally, pill zonescontaining dry reagents pills are located on cover layer 1332 in theregions that are exposed by openings 1345 and 1346 in gasket 1330 sothat they the reagents are reconstituted in liquids passing through thepill zones on the way to detection chambers 945 and 946. Cover layer1321 seals air chamber/trap 976 and the top side conduit segments whichinclude double z-transition connecting segments 1070 and 1071. Coverlayer 1320 seals sample introduction port 921 and reagent introductionport 922.

In the preferred embodiment shown in FIGS. 11 and 13, the cartridge bodyfurther includes electrical access regions 995 and 996 that, togetherwith cutouts 1370 and 1371 in gasket layer 1331 allow electrical contactto be made with electrode contacts 997,998. Electrical access regionsare cut-outs or holes in the cartridge body configured and arranged tobe in alignment with the electrode contacts.

At least a portion of cartridge body 1100 is adapted and configured tobe an optical detection window and is arranged in optical registrationwith the electrodes to allow optical detection of luminescence generatedby the electrode array. In one particularly preferred embodiment, thecartridge body and/or the cover layers are fabricated from a translucentmaterial. The use of optically transparent materials has the furtheradvantage that optical detectors, e.g., detectors arranged within acartridge reader, can be used to detect the presence of liquids in theconduits. These optical detectors can be used to ensure that thecartridge is functioning properly and to provide feedback to the controlsystems controlling fluid movement in the cartridge. Alternatively, thecartridge body and/or cover layers may contain optical detection windowsthat are properly arranged locations that require optical detection offluid presence and/or composition (e.g., detection ofreflectance/transmittance from a light source). FIG. 12 depictspreferred locations for optical detection points 1210-1217 in cartridge900.

FIG. 14 a is a schematic representation of the fluidic components ofcartridge 1400, another preferred embodiment of the cartridge of theinvention. FIGS. 14 b and 14 c show exploded views of one preferreddesign of cartridge 1400. FIG. 18 is a three dimensional representationof the fluidic network of this design. Cartridge 1400 comprises a samplechamber 1420, first and second reagent chambers 1425 and 1426, detectionchambers 1445 and 1446, waste chambers 1430 and 1431. Sample chamber1420 is preferably adapted to receive a liquid sample and is linked viavent conduit 1475 to vent port 1480 and via sample conduit 1415(including sample conduit branches 1440 and 1441 that branch fromdistribution point 1540) to detection chambers 1445 and 1446. Ventconduit preferably has a serpentine shape to increase its length andprevent fluid from bubbles in sample chamber 1420 from back-flowing intovent port 1480. Sample conduit 1415 preferably comprises a z-transitionnear the conduit connection to the sample chamber 1420 for preventingpremature leakage of sample from sample chamber 1420. Sample chamber1420 also has sample introduction port 1416 and cap insert 1414 forsealing the port. Optionally, sample conduit branches 1440 and/or 1441comprise reagent pill zones.

Reagent chambers 1425 and 1426 are, preferably, adapted to hold reagentampoules. Reagent chamber 1425 is connected via a reagent vent conduitto vent port 1450 and via reagent conduit 1470 to sample conduit 1415.Reagent conduit 1470 is further connected via vent conduit 1482 to ventport 1481 which may be used to introduce air into reagent conduit 1470and downstream conduits such as sample conduit branches 1440 and 1441.Advantageously, reagent conduit 1470 has an extended segment betweenvent conduit 1482 and sample conduit 1415 which may be used as a stagingarea for a defined volume of liquid reagent. Preferably, this extendedsegment also comprises a reagent pill zone for introducing a dry reagentinto the liquid reagent held in reagent chamber 1425. Reagent chamber1426 is connected via a vent conduit to vent port 1451 and via reagentconduit 1427 to sample conduit 1415 (first intersecting with reagentconduit 1470 just downstream from sample conduit 1415). Reagent conduits1427 and 1470 preferably comprise Z-transitions near to the connectionof the conduits to their corresponding reagent chambers to preventpremature leakage of the reagent from the chambers. Detection chambers1445 and 1446 preferably, comprise immobilized binding reagents foranalytes of interest, preferably an array of binding reagents,preferably an array of binding reagents supported on electrode arraysfor conducting ECL measurements, e.g., the electrode arrays of theinvention as described above. Detection chambers 1445 and 1446 connectto sample conduit branches 1440 and 1441 and to waste conduits 1460 and1461. Waste chambers 1430 and 1431 connect to waste conduits 1460 and1461 and, via vent conduits to vent ports 1452 and 1453. Optionally, onedetection chamber (and the associated fluidics and waste chamber) may beomitted.

Cartridge 1400 is adapted to carry out one and two step washed assays(assays that involve treating a detection chamber with one or twosamples/reagents prior to conducting a wash step). A preferredembodiment of a one step washed assay comprises: i) introducing samplefrom sample chamber 1420 into detection chambers 1445 and/or 1446 viasample conduit branches 1440 and/or 1441 (optionally, the sampleintroduced into the detection chambers including reconstituted reagentssuch as labeled binding reagents and/or control/calibration reagentspicked up in pill zones comprised in sample conduit branches 1440 and/or1441) ii) washing detection chambers with a wash reagent contained inreagent chamber 1426 (the reagent preferably comprising anelectrochemiluminescence coreactant and providing a suitable environmentfor an ECL measurement) and iii) interrogating the contents of thedetection chamber (preferably, by conducting an ECL measurement). Forcartridges carrying out such a one step protocol, reagent chamber 1425may be omitted (in which case, vent port 1481 may be directly connectedto reagent conduit 1427 or sample conduit 1415. A preferred embodimentof a two-step washed assay comprises: i) introducing sample from samplechamber 1420 into detection chambers 1445 and/or 1446 via sample conduitbranches 1440 an and/or 1441 (optionally, the sample introduced into thedetection chambers including reconstituted reagents such as blockingagents, buffers, labeled binding reagents and/or control/calibrationreagents picked up in pill zones comprised in sample conduit branches1440 and/or 1441); ii) introducing a liquid reagent from reagent chamber1425 into detection chambers 1445 and/or 1446 (optionally, the reagentintroduced into the detection chambers including reconstituted reagentssuch as blocking agents, buffers, labeled binding reagents and/orcontrol/calibration reagents picked up in pill zones comprised inreagent conduit 1470); iii) washing detection chambers with a washreagent contained in reagent chamber 1426 (the reagent preferablycomprising an electrochemiluminescence coreactant and providing asuitable environment for an ECL measurement) and iv) interrogating thecontents of the detection chamber (preferably, by conducting an ECLmeasurement). Optionally, a wash step is included between steps (i) and(ii). Advantageously, the use of a two step format in binding assaysallow analyte or other components in a sample to be bound to immobilizedbinding reagents in the detection chambers and washed out of thedetection chamber prior to the introduction of labeled detectionreagents (e.g., labeled binding reagents for use in sandwich bindingassays or labeled analytes for use in competitive assays); carrying outassays in two steps may be advantageous in competitive assays and assaysthat suffer from large sample matrix effects or hook effects. Someassays may not require a wash step (e.g., non-washed ECL assays may becarried out by incorporating adding an ECL coreactant to the sample);for cartridges carrying out such non-washed assays (in one or two stepformats), reagent chamber 1426 may be omitted.

A shown in FIG. 14 b, a preferred embodiment of cartridge 1400 uses alaminar cartridge design employing a two part cartridge body (1410 and1411) and cover layers 1401, 1402, 1403 and 1407. To allow for adequatesample and/or reagent volumes, the cartridge body has a thicker portionwhich includes features (channels, grooves, wells, compartments, etc.)that define, in part, the sample, reagent and waste chambers. Theremainder of the cartridge is, preferably, much thinner so as tominimize cartridge weight, volume and material costs. The two partcartridge design is not required but is advantageous for producing thecartridge by low cost injection molding techniques by allowing thethicker regions of the cartridge body to be hollowed out thus reducingthe amount of material needed to produce a cartridge, reducing the timerequired to cool the parts before ejection from an injection mold dieand reducing the part deformation after release from the mold. In thishollowed out design, through-holes through the cartridge body can beprovided for by tubes incorporated into body components 1410 and/or 1411(see, e.g., tube 1439 in FIG. 14 b). These tubes may be mated to tubesor holes in the other body component to form through-holes through thebody. This mating can be accomplished by a variety of methods includingtube mating methods known in the art. Preferred techniques includeplastic welding techniques and/or the use of press fits (preferably, bymating a tapered tube with an outer diameter that decreases from d_(max)to d_(min) at its end with a tube that has an inner diameter betweend_(max) and d_(min)). In an alternate embodiment, a one part cartridgebody is used.

At least portions of the sample, reagent and vent conduits are formed bysealing cover 1403 on lower cartridge body part 1410. Detection chambers1445 and 1446, portions of sample conduit branches 1440 and 1441, andportions of elongated reagent conduit 1470 are formed by sealing coverlayer 1407 (having patterned conductive layer 1423 (which forms apatterned electrode array analogous to the electrode array 963, shown inFIG. 9) and patterned dielectric overlayers 1421,1422) to lowercartridge body part 1410 through intervening gasket layer 1405(preferably, made from double sided adhesive tape). The detectionchamber's depth, length and width are defined by cutouts 1447 and 1448within the gasket layer. Cutouts 1406,1408,1412,1413 in the gasket layerexpose regions of dielectric layers 1421 and 1422 to sample conduitbranches 1440 and 1441 and elongated reagent conduit 1470.Advantageously, dry reagent pills comprised within these reagents arelocated on these regions. This choice of pill locations allows dryreagent pills and/or immobilized reagents within the detection chambersto be dispensed on a single substrate. Preferably, as shown in FIG. 14,sample conduit branches 1440 and 1441 have segments that are adjacentand/or substantially parallel to detection chambers 1445 and 1446 and aU-turn segment to allow connection to the detection chambers. Thisarrangement provides for conduit lengths that are long enough to allowfor the introduction of a sample to the conduit and mixing of the samplewith a pill in the conduit prior to introduction of the sample to thedetection chamber. These lengths are achieved without adding to thelength of the cartridge. Advantageously, this arrangement also allowsthe patterned electrode layer to be used to conduct capacitive orconductometric measurements of fluid within the sample conduits asdescribed above. Similarly, elongated reagent conduit 1470 has entranceand return segments, connected via a U-turn segment, that are parallelto detection chambers 1445 and 1446. Lower cartridge body component 1410further includes electrical access regions 1432 and 1433 that, togetherwith cutouts 1417 and 1418 in gasket layer 1405 allow electrical contactto be made with conductive layer 1423.

Cover layer 1402 mates to lower cartridge body component 1410 to defineconduit segments 1805 (readily seen in FIG. 18 a) that (by connectingtwo z-transitions) act as bridge segments connecting the fluidicnetworks defined by cover layers 1403 and 1407. Optionally, pill zonesformed on cover layer 1402 on surfaces of bridge segments comprisedwithin the sample or reagent conduits may be used to introduce dryreagents to the sample or liquid reagents. Cover layer 1401 mates toupper cartridge body component 1411 and seals reagent chambers 1425 and1426, preventing the release of fluid from ampoules within the chambers.Cover layer 1401 also seals top side conduit segments including doublez-transition connecting segments such as segments 1810 and 1815 readilyseen in FIG. 18 a.

FIG. 15 a shows a top view of upper body component 1411. FIGS. 16 a and16 b show top and bottom views of lower body component 1410. As shown inFIG. 15 a, the upper cartridge component 1411 preferably includesreagent chambers 1425,1426 that are configured to hold reagent ampoules.Filters 1515,1516 are preferably integrally molded into the uppercartridge component to ensure that substantially all of the glassfragments from the ruptured glass ampoules are not permitted to enterthe fluidic network and possibly obstruct/block fluid flow.Alternatively, the filters may be separate components that areincorporated into the sample and/or assay reagent chambers during themanufacturing/assembly process; e.g., inserts that may preferably besnapped into place (see, e.g., inserts 2020 and 2021 in FIG. 20).

The two piece cartridge design also advantageously simplifies theemployment of additional anti-foaming measures in the waste chambers. Avertical web, or partial wall, can be included in the upper portions ofthe waste chambers 1610,1611 located in the upper cartridge component1600, another embodiment of upper cartridge component 1411. Preferablythe anti-foaming web is arranged between the waste chamber vent and thewaste chamber input. The height of the anti-foaming web preferablyextends the full depth of the upper portion of the waste chamber but maybe less than the full depth as well. Alternatively, the anti-foaming webcan extend beyond the depth of the upper portion of the waste chamber sothat it protrudes into the lower portion of the waste chamber.Preferably the height of the anti-foaming web is selected to achieveoptimum anti-foaming.

As discussed above, the input conduits of the waste chambers arepreferably arranged so as to enter the waste chambers in a manner thatallows the waste fluid to run down the wall of the waste chamber tominimize or eliminate foaming. As illustrated in FIG. 16 a, the inputconduits 1615,1616 intersect one of the walls of the waste chambers.Additionally, the vents are configured and arranged to access the wastechambers at a point that will be above the anticipated fluid level.Locating the waste chamber vents at or near the top of the waste chamberalso helps to ensure that any foaming that may occur within the chamberdoes not result in fluid entering the vent line and possiblycontaminating the cartridge reader instrument.

FIG. 32 shows a schematic of the fluidic network of cartridge 3200, apreferred embodiment of the invention configured to extract analyte froma matrix, preferably from an applicator stick, most preferably from aswab. FIG. 33 shows an exploded view of a preferred design of cartridge3200. Cartridge 3200 illustrates two preferred features of cartridges ofthe invention: a sample chamber for extracting analyte from a matrix andthe use of a “reverse flow” wash. Cartridge 3200 has reagent chamber3210 linked to vent port 3212 and extraction reagent conduit 3214preferably, comprising a Z-transition). Reagent chamber 3210 holds aliquid reagent suitable for extracting the analyte. Preferably, reagentchamber holds an ampoule of nitrous acid or, more preferably, an ampouleof an acid (preferably, acetic acid) and a dry nitrate salt outside ofthe ampoule so that rupturing the ampoule leads to the formation ofnitrous acid. Nitrous acid is a particularly useful extraction reagentfor extracting cell wall antigens from gram positive bacteria and mayalso be used to extract markers from other organisms in mucus containingsamples such as upper respiratory samples (see, e.g., the extractionmethods and reagents disclosed in U.S. Provisional Patent Application60/436,591, filed Dec. 26, 2002, entitled Methods Compositions and Kitsfor Biomarker Extraction, hereby incorporated by reference).

Cartridge 3200 has elongated sample chamber 3220 (a sample chamberconfigured for extracting samples such as those described above inconnection with FIGS. 28-30) connected to extraction reagent conduit3214 and sample conduit 3224 so as to allow the flow of extractionreagent through the sample (preferably, through swab head 3205).Preferably, as shown in FIG. 33, sample chamber 3220 is angled or curvedalong its elongated dimension so as to aid in breaking a scored swabinserted into the sample compartment. Sample conduit 3224 is connectedto bubble trap 3226 (preferably connected to bubble trap vent port 3266)for removing air from the extracted sample and waste chamber 3228 (whichis preferably connected to waste vent port 3262). Further downstream,sample conduit 3224 is connected to detection chamber 3230. Sampleconduit 3224 comprises pill zone 3225 which may hold labeled bindingreagents (e.g., labeled antibodies for use as detection reagents insandwich immunoassays) and/or a neutralization reagent (e.g., a pHbuffering component such as Tris, Hepes, phosphate and the like) forneutralizing an acidic extraction reagent in the sample (such as nitrousacid).

Detection chamber 3230, preferably, comprises immobilized bindingreagents for analytes of interest, preferably an array of bindingreagents, preferably an array of binding reagents supported on electrodearrays for conducting ECL measurements as described for other cartridgeembodiments above. In an especially preferred embodiment the bindingreagents are antibodies directed against markers of organisms(preferably including at least one gram positive bacteria, mostpreferably a Streptococcus species) that may be found inmucus-containing sample such as upper respiratory samples (see, e.g.,the organisms described in U.S. Provisional Patent Application60/436,591, filed Dec. 26, 2002, entitled Methods Compositions and Kitsfor Biomarker Extraction, hereby incorporated by reference). Detectionchamber 3230 is connected to wash reagent chamber 3240 via wash reagentconduit 3242 (which, preferably, comprises a Z-transition). Vent port3244 is arranged along wash reagent conduit 3242 between detectionchamber 3230 and wash reagent chamber 3240. Wash reagent chamber 3240 isalso connected to vent port 3241. Wash reagent chamber 3240 comprises aliquid wash reagent, preferably in an ampoule. The liquid was reagent,preferably, comprises an ECL coreactant and provides an appropriatechemical environment for an ECL measurement.

The fluidic arrangement of cartridge 3200 allows for forward flow ofextracted sample through pill zone 3225 into detection chamber 3230 andreverse flow of sample into waste chamber 3228 and wash reagent fromwash reagent chamber 3240 into detection chamber 3230.

Cartridge 3200 also has optional control detection chamber 3250 which ispreferably configured like detection chamber 3230. The fluidicarrangement of the cartridge allows wash reagent from wash reagentchamber 3240 to pass through pill zone 3252 to detection chamber 3250.Pill zone 3252, preferably, comprises the same binding reagents as pillzone 3225 but also comprises control reagents (preferably, predeterminedamount of the analytes measured in detection chamber 3230) so thatreconstitution with wash reagent forms a control sample. The fluidicarrangement further allows the forward flow of control sample into wastechamber 3254 (which is preferably connected to waste vent port 3264) andwash reagent from wash reagent chamber 3240 into detection chamber 3250.

As shown in FIGS. 32 and 33, cartridge 3200, preferably, employs many ofthe same design features as preferred embodiments of cartridge 900and/or 1400 such as use Z-transitions, laminar construction, electrodearrays, bridge segments, and the like. As shown in FIG. 33, cartridge3300, preferably, has a two part design. Advantageously, this designallows the sample chamber to be constructed from two sections andsimplifies the manufacture of the curved/angled elongated chamber. Asshown in FIG. 33, cartridge 3200 may also comprises a bar code 3295 orother identifying feature that can, e.g., identify the assay panelcarried out on the cartridge, the cartridge lot, the time ofmanufacture, the expiration date, cartridge specific calibration data,the sample source, etc.

The fluidic components are preferably adapted and configured to form afluidic system that can be selectively controlled via a cartridge readerinstrument. The cartridge reader 2300 is schematically depicted in FIG.23 and preferably incorporates various subsystems for performing thepredetermined assay. The cartridge reader is shown holding a cartridge2390 which may be supplied separately. As depicted, the cartridge readerpreferably includes the cartridge handler 2315, the fluidic handler 2340and the assay electronics 2330 subsystems. Together these subsystems arepreferably controlled by an electronic control system 2310 responsible,generally, for directing the cartridge handler subsystem to load andposition the cartridge within the reader, for controlling/coordinatingthe introduction/movement of fluids throughout the fluidic network andfor directing the assay electronics to perform the assay measurement.The cartridge reader is preferably packaged as a single self-containedunit. In preferred embodiments employing luminescence based assays, asmaller light-tight region is incorporated within the overall cartridgereader housing. This allows the luminescence based assay to be performedwithin the light tight enclosure to ensure that the readings are notaffected by ambient light. Preferably, electronic components and otherheat-generating components are located outside of the light tightenclosure.

The cartridge handler subsystem preferably includes a motor to draw thecartridge into the cartridge housing and selectively position thecartridge within the cartridge reader; e.g., position the cartridgeunder a sensor/detector 2335. In one preferred embodiment, retraction ofthe cartridge within the cartridge reader housing may be mechanicallycoupled to one or more mechanisms within the cartridge reader forsynchronized/coordinated operation of the linked mechanisms. Forexample, the retraction of the cartridge may be mechanically coupled to:the mechanism for closing the door 2325 to the light tight enclosureafter the cartridge has entered the chamber; the assay electronicssubsystem (described in greater detail below) to allow the cartridgereader's electrical contacts 2330 to engage the cartridge's electricalcontacts, i.e., be placed into electrical contact with the electrodearray's electrode contacts; the fluidic handler subsystem's (describedin greater detail below) fluidic manifold 2340 to engage the cartridge'sfluid ports, i.e., be placed into fluidic communication with thecartridge's fluidic ports (e.g., establishing a pressure seal betweenthe cartridge's fluidic ports and the fluid manifold); and/or the fluidhandler subsystem's reagent module breaking mechanism 2350 to allow thereagent modules such as ampoule(s) to be broken during the cartridgeretraction/positioning step.

In certain embodiments the measurement step may comprise reading thesignal from each read chamber separately. While this may be accomplishedby using a single suitable detector and optimal positioning of thecartridge's read chambers in relation to the single detector, successfulmeasurement/detection may also be carried out by repositioning thedesired read chamber in relation to the single detector or repositioningthe detector in relation to the desired read chamber. For such anembodiment, the cartridge handler subsystem may include a separate motorto allow for positioning of the cartridge and/or the detector. In aparticularly preferred embodiment, the cartridge handler subsystem isadapted and configured to precisely position the cartridge or thedetector, or both, such that the detector is in registered alignmentwith the precise location where the measurement is being performed;e.g., the working electrode presently being stimulated to produce ECL.

In a preferred embodiment a barcode reader 2365 is incorporatedon/within the cartridge reader to preferably automatically scan anidentifying mark/label 2370 on the cartridge; e.g., as it is drawn intothe reader. The label may contain encoded information relating to thespecific assays that are to be performed, calibration parameters and/orany other information required to perform the assay. Further, apreferred embodiment may incorporate a heater within the cartridgereader to warm the cartridge to a predetermined temperature, e.g., 37°C., before proceeding.

Preferably, the reader does not come in contact with liquids containedwithin the cartridge. This feature may be accomplished by usingpneumatic pressure applied at the vent ports to drive fluids in thecartridge. The fluidic handler subsystem preferably includes a pump 2345(preferably a piston pump) to selectively apply positive and/or negativepressure (i.e., apply a vacuum) to one or more of the cartridge'sfluidic components in order to selectively control movement of fluidswithin, and through, the cartridge and its various fluidic components.The fluidic handler subsystem is preferably adapted and configured tofluidically engage the cartridge at one or more fluidic control points;e.g., positive control ports, vent ports, and the like and includesfluidic connectors for providing these fluidic engagements. Selectiveapplication of pressure to the cartridge's fluidic components ispreferably achieved by incorporating a fluid manifold 2340 housed withinthe cartridge reader to simplify and enhance the fluidic engagementfunction and to minimize the number and complexity of fluidic systems.Advantageously, the fluidic manifold 2340 can be adapted and configuredto facilitate the use of a single pump; i.e., control valves 2342 can beincorporated within the fluidic manifold 2340 to selectively controlfluid movement within and through the various fluidic components of thecartridge. The fluidic handler preferably includes a pressure sensor tofacilitate precise/repeatable movement and/or positioning of fluidswithin the fluid network. The fluidic connectors, preferably, compriseaerosol-prevention plugs or gas-selective membranes (i.e., materialsthat selectively allow the passage of gas but prevent the passage ofliquids) to prevent contamination of the reader fluidics with liquids ina cartridge. The components comprising these plugs or membranes are,preferably, easily removed and replaced if they become contaminated withliquid. Aerosol-prevention plugs are commonly used in pipette tips toprevent contamination of pipettors and include materials that allow thepassage of air when dry but swell and seal up the passage when they comein contact with liquid (e.g., filter materials impregnated or coatedwith cellulose gum).

The fluidic handler subsystem preferably employs fluid sensors (notreadily seen in FIG. 23. FIGS. 12 and 17 illustrate alternative fluidsensor layouts in relative arrangement to the cartridge/fluidicnetwork), e.g., reflective photo sensors, positioned at predeterminedlocations within the fluid network; In accordance with these preferredembodiments, the fluid sensors are positioned in registered alignmentwith the labeled optical detection points located on the cartridge body.Sensor signal data may be used to provide fluid positional informationwhich may be used to control pump operational parameters such as pumpspeed, direction and the duration of a specific pump operation. Inaddition to precise control of fluid movement within and throughout thecartridge, fluid sensors may be used to control mixing of fluids (e.g.,during the incubation period, and evacuation of sample from the readchambers during the wash and read cycle) by, e.g., defining the limitsof the motion of slug fluid fronts during back and forth mixing motionsand/or by measuring an optical property of the fluid such as absorbanceor light scattering that is indicative of the state of a mixingoperation. The fluid sensors may also be used to conduct viscositymeasurements on a sample. In one embodiment, the reader pump is directedto move the fluid front of a sample through a fluidic conduit from oneoptical sensor position to another by operating the pump at a predefinedspeed or under conditions designed to achieve a predefined pressuregradient. The time needed to move the fluid between the two positions isindicative of the viscosity. Such a viscosity measurement is optionallyused to measure the coagulation time of a blood or plasma sample (e.g.,whole blood clotting time, thrombin time, prothrombin time, partialthromboplastin time and/or activated clotting time). Such a method mayfurther comprise introducing one or more coagulation reagents (e.g., bypassing the sample over a dry reagent comprising these reagents) priorto conducting the timing step. Suitable reagents for measuring thrombintime may include thrombin. Suitable reagents for measuring prothrombintime may include thromboplastin and/or calcium. Suitable reagents formeasuring partial thromboplastin time may include cephalin and anegatively charge substance (preferably, diatomaceous earth, kaolin,glass particles and/or ellagic acid). Suitable reagents for measuringactivated clotting time may include negatively charged substances suchas diatomaceous earth, kaolin, glass particles and/or ellagic acid.

While the use of optical sensors to monitor fluid flow is advantageous,it is not required. In certain alternate embodiments, fluid movementoperations are conducted by operating a pump for a predefined time atpredefined speeds, or under conditions which have been determined (e.g.,through calibration of the pump) to result in a predetermined movementof a fluid slug.

The assay electronics subsystem preferably includes electrical contacts,sensors and electronic circuitry. The electrical contacts 2330 arepreferably adapted and configured to be placed into electrical contactwith the electrode array. In one preferred embodiment, the cartridgereader's electronic circuitry may include analog switching andtrans-impedance amplification circuits to address a specific pair ofelectrodes (i.e., pair-wise firing, discussed in greater detail above)and apply a predefined voltage waveform to the circuit formed by thatelectrode pair. The actual output voltage and current may be optionallymeasured for diagnostic purposes. Preferably the electronic circuitry isalso capable of applying an AC waveform (e.g., 500 Hz or less) forcapacitive or conductive measurements (as discussed above). Stillfurther, the electronic circuitry may be configured to generate 20 kHzsignals suitable for, e.g., hematocrit measurements of blood samples.

In one particularly preferred embodiment of the cartridge readerconfigured to perform luminescence based assays, the cartridge readermay employ an optical detector 2335, e.g., a photodiode (mostpreferably, a cooled photodiode), photomultiplier tube, CCD detector,CMOS detector or the like, to detect and/or measure light/luminescenceemanating from the read chambers. If a cooled photodiode is employed, athermo-electric cooler and temperature sensor can be integrated into thephotodiode package itself providing for selective control by theelectronic control system.

A computerized control system 2310 is preferably utilized to selectivelycontrol operation of the cartridge-based system. The computerizedcontrol system may be fully integrated within the cartridge reader,separated from the cartridge reader in an externally housed systemand/or partially integrated within and partially separated from, thecartridge reader. For example, the cartridge reader can be configuredwith external communications ports (e.g., RS-232, parallel, USB, IEEE1394, and the like) for connection to a general purpose computer system(not shown) that is preferably programmed to control the cartridgereader and/or its subsystems. In one preferred embodiment, a singleembedded microprocessor may be used to control the electronics and tocoordinate cartridge operations. Additionally, the microprocessor mayalso support an embedded operator interface, connectivity and datamanagement operations. The embedded operator interface can preferablyutilize an integrated display 2360 and/or integrated data entry device2355 (e.g., keypad). The computerized control system may also preferablyinclude non-volatile memory storage for storing cartridge results andinstrument configuration parameters.

FIG. 34 shows a cutaway exploded view of one preferred design for reader2300 and also shows a cartridge drawer 2386 (preferably comprising anintegrated cartridge heater) on linear guide 2384 and driven by motor2380 for moving the cartridge in and out of the reader. FIG. 34 alsoshows fluid sensor array 2388 (holding sensors, preferably optical) fordetecting fluid at selected positions in the cartridge and a motor 2382for bringing the cartridge together with frame 2383 which supports theelectrical connectors (not shown in this view), fluidic connectors (notshown in this view), ampoule breaking mechanism 2350 and light detector2335.

FIG. 24 illustrates a preferred configuration of valves in a cartridgereader fluidic handling sub-system configured for use with cartridge2500 (analogous to cartridge 1400) shown in the fluidic diagram of FIG.25 (along with preferred locations for cartridge reader fluid detectionsensors 1-15). The sub-system comprises a pumping system that comprisesa pneumatic pump (preferably, an air piston) linked to a pump manifold.The manifold is connected to control lines (comprising control valves2412A and 2412B) that connect the pump to selected vent ports(preferably, the waste chamber A vent port 2512A and waste chamber Bvent port 2512B) on a cartridge and allow the pump to be used to movefluid in the cartridge away or towards the selected vent ports. Themanifold is also connected to a pump vent line (comprising a pump ventline valve 2492) for venting the pump manifold. The control valves havea closed position that seals the control line and the associatedcartridge vent port, an open position that connects the pump to thecartridge vent port and, optionally, a vent position that opens thecartridge vent port to ambient pressure. The pump vent line valve has aclosed position that seals the pump vent port and an open position thatexposes the pump manifold to ambient pressure and releasespressure/vacuum in the pump manifold. The fluidic handling sub-systemfurther comprises vent lines (comprising vent valves 2412, 2422, 2432Aand 2432B) that allow venting of vent ports (sample chamber vent port2512, air port 2522, reagent chamber A vent port 2532A and reagentchamber B vent port 2532B, respectively) on a cartridge (preferably, thecartridge vent ports other than the waste cartridge ports). The ventvalves have a closed position that seals the associated cartridge ventport and an open position that exposes the vent port to ambientpressure. The fluidic handling sub-system may also comprise a pressuresensor couple to the pump manifold for detecting pressure in themanifold. During fluidic control of a cartridge, the pressure in themanifold is, preferably, monitored to ensure that it falls withinexpected pressure ranges for specific operations and confirm that thefluidic handling system is operating properly. The specific preferredvalve configuration shown in FIG. 24 is designed to move fluid primarilyby aspirating it towards the valve chambers. Other valve configurations,e.g., configurations that drive fluids primarily by positive pressure,will be readily apparent to the skilled artisan and may valves thatallow chambers other than the waste chambers to be connected to the pumpand/or that allow the waste chambers to be directly vented to theatmosphere.

With reference to FIGS. 24 through 26, performance of an assay using apreferred cartridge of the invention will be described. This exemplarymethod will be described in the context of a two-step multiplexedbinding assay using antibodies as binding reagents and ECL as thedetection methodology, however, it will be readily apparent to theskilled practitioner that the described fluidic operations can be usedin a variety of different assay formats (e.g., binding assays usingother classes of binding reagents, enzymatic assays, etc.) and with avariety of different detection technologies. It is also apparent thatthe sequence of operation discussed below may vary according todifferences in the configuration of a particular cartridge as well asdifferences in the particular assay to be performed.

During operation, the pump vent line valve may be used to enable anddisable pressurization of the system for more precise fluid control;when the pump's vent is opened, the system returns to ambient pressurevery quickly. Typical fluid draw operations, i.e., routing of fluidwithin and throughout the fluid network, involve closing the pump ventvalve and opening i) one or more (preferably, one) cartridge ventvalves, e.g., the sample, air, reagent chamber A and/or reagent chamberB vent valves and ii) one or more (preferably, one) control valves,e.g., waste chamber A or waste chamber B control valves. Therefore, aslug of fluid will move along a path through the fluid network in thecartridge when the fluid channels comprising that path is vented to airat one end and subjected to either pressure or vacuum at the other end.

A user selects the appropriate cartridge for carrying out a desiredmeasurement and introduces sample to the sample introduction port of acartridge and, preferably, seals a closure on the sample introductionport. The cartridge is inserted into the cartridge reader. Preferably,the cartridge will include features that ensure the cartridge isinserted in the proper orientation; e.g., by incorporating identifyingmarks to show which direction it should be placed on the tray and/ormechanical features that guide the user to place it in the correctorientation. After the user has successfully prepared and inserted thecartridge, reading/processing of the cartridge is performed by thecartridge reader upon receiving an indication from the user that theread cycle should commence (alternatively, the reader may automaticallybegin operation upon confirming that a properly prepared cartridge hasbeen properly inserted into the cartridge reader). The subsequentreading of the cartridge is preferably automated; e.g., the cartridgereader's electronic control system (computerized control system or thelike) automatically processes and reads the cartridge.

The automated sequence of operations to be performed by the cartridgereader will now be described. Preferably the cartridge includes machinereadable indicia, e.g., barcode, that is detected and processed by thecartridge reader. For example, processing of the machine readableindicia may allow the cartridge reader to verify that a valid, readablebarcode has been detected and thereafter determine the operationalparameters for the present read cycle; i.e., determine the set ofassays/tests to be performed, extract any relevant instrumentconfiguration parameters and verify the expiration date. In certainpreferred embodiments, the cartridge reader may prompt the user for anydata that it requires; e.g., operator ID, sample or patient ID, and thelike. Additionally, if the cartridge is capable of running a panel oftest, the user may be able to select which test(s) within the panelshould want be performed.

Preferably, the reader has a cartridge handling subs-system thatmechanically engages the cartridges and moves/aligns it into position.Preferably, this process includes positioning the cartridge within alight-tight enclosure. The reader also makes the appropriate fluidicand/or electronic connections to the cartridge and, optionally, breaksor pierces any reagent modules (e.g., reagent ampoules) present incartridge reagent chambers. As discussed above, in one preferredembodiment, the cartridge handler's motion would be physically coupledto the fluidic and electronic handlers (and, optionally, the reagentmodule release mechanism) such that upon positioning the cartridgewithin the light tight enclosure the electrical contacts and thefluidics manifold engage the cartridge at their respective engagementpoints (and, optionally, the reagent module release mechanisms releasesreagent from any reagent modules). Next, where required or preferred,the electronic control system begins operating a heater in order tobring the cartridge to the appropriate predetermined temperature andmaintain the cartridge at such target temperature. In certain preferredembodiments temperature regulation may be controlled by a microprocessoremploying a proportional derivative control to control a heater thatwill maintain the target temperature; preferably a suitable algorithm isemployed.

Once the cartridge has been maintained at the target temperature for apredetermined amount of time, the fluid handler may begin processing thecartridge for reading; i.e., assemble the assay. Reference to FIG. 26will be made to illustrate the intermediary states of the cartridgereader and the position of fluid within the fluid network of cartridge2500 during a 2-step assay format. As presented in FIG. 26, the startingstate of the cartridge 2500 (panel 2601) is illustrated and depicts thelocation of the constituent fluids within the fluidic network. Assayassembly preferably consists of metering specific volumes of samplefluid, reconstituting dried reagents in the sample fluid and incubatingthe sample fluid in the detection chambers. Predetermined valves areopened in a prescribed sequence in accordance with the desired fluidflow paths to be assumed by the constituent fluids.

According to the present embodiment in which two read chambers arepresent and will be utilized for testing the sample, two equal lengthsof sample fluid (i.e., slugs) will be drawn; the length of the sampleslugs is determined by the volume of the read chambers. The sample slugsare delimited from one another by introducing a slug of air between thetwo sample slugs. Accordingly, sample chamber vent valve 2412 and awaste chamber vent valve 2442A are opened and the pump vent is closed.The pump is subsequently activated to aspirate/draw the sample fromsample chamber 2510 (preferably, overcoming a capillary break providedby a Z-transition that is used to prevent leakage of the sample from thesample chamber) into sample conduit branch 2515A. In this and otherpumping steps, a pressure sensor (not shown), preferably, detects thepressure created by the operation and provides confirmation that thepump is aspirating/dispensing fluid properly. When fluid is detected atsensor 3 (see FIG. 26, 2602), the pump vent valve is opened and the pumpmotor is deactivated. The sample chamber vent valve 2412 and wastechamber vent valve 2442A are then closed. Similarly, sample is drawninto sample conduit branch 2515B by operating the pump with samplechamber vent valve 2412 and waste chamber B vent valve 2442B open (seeFIG. 26, panel 2603). Defined slugs of sample fluid are drawn into thesample conduit branches by operating the pump with air vent valve 2422open as well as the waste chamber A and B vent valves 2442A-B (see FIG.26, panel 2604). In this and subsequent steps, two slugs may be movedsimultaneously through sample conduit branches 2515A and B by holdingboth waste chamber vent valves open or sequentially through the branchesby opening one at a time.

The sample conduit branches, preferably, comprise dry reagent pills(preferably containing one or reagents selected from blocking agents, pHbuffers, salts, labeled binding reagents, and the like). One or more ofthe conduit branches may also comprise spiked analyte for spike recoverycontrols. In order to reconstitute the dried reagent, the two samplefluid slugs are moved back and forth across the pill zone apredetermined number of times by opening air vent valve 2422 and wastechamber vent valves 2442A and/or B and operating the pump to alternatebetween applying positive and negative pressure to the waste chambervents (FIG. 26, panels 2605-2606). The two sample fluid slugs may bemoved back and forth simultaneously or mixing of the two slugs may beaccomplished in series. The number of repetitions that the sample fluidis cycled across the pill zone may be dependent upon a number offactors, including but not limited to, size/volume of reagent driedreagent pill, composition of reagent pill, drying method employed at thetime of reagent deposition/pill formation, and the like. In accordancewith preferred embodiments, the number of repetitions that need to becarried out by the fluid handler subsystem can be cartridge specific andcan be automatically ascertained by the cartridge reader from theinformation encoded in the machine-readable indicia affixed/incorporatedonto the cartridge. The number of repetitions may be predeterminedthrough empirical results but may also be determined in-situ through theuse of one or more sensors adapted and configured to measure the degreeof mixing of the reagent(s) and sample fluid; e.g., use of opticalsensors (transmittance or reflectance), electrical sensors (impedance,conductance, resistance, and the like).

The sample fluid slugs are now moved into their detection chambers 2550Aand 2550B by operating the pump with air vent valve 2422 and wastechamber vent valve 2442A open until the sample slug is detected atsensor 7 and by operating the pump with air vent valve 2422 and wastechamber vent valve 2442B open until the sample slug is detected atsensor 8 (FIG. 26, panels 2607-2608). The sample slugs are incubated inthe detection chambers to allow constituents of the sample (e.g.,labeled binding reagents, analyte, control analyte, etc.) andimmobilized binding reagents within the detection chamber to bind toform binding complexes in the detection chamber. Preferably, a mixingoperation is employed to enhance the rate of these binding reactions.Preferably, mixing is achieved by moving the fluid slugs back and forthin the detection chamber by a process analogous to that described forreconstituting the reagent pill (optionally, using sensors 1, 2, 11 and12 to provide stopping points in each direction). The aspirate anddispense operations are repeated a predetermined number of times, oruntil the degree of mixing desired has been achieved/detected. Aftercompletion of the incubation step, the air and waste chamber vent valvesare used to draw the slugs out of the detection chambers and into wastechambers 2540A and B (FIG. 26, panels 2609-2610).

Preferably (as shown), the assay process includes a wash step forremoving sample and unbound labeled reagents from the detection chamber.The wash uses a wash reagent (preferably, a buffered solution, morepreferably comprising a non-ionic surfactant such as Triton X-100 andmost preferably comprising an ECL coreactant such as TPA or PIPES)stored in reagent chamber A 2530A. If the wash reagent is in a reagentmodule (preferably, ampoule) and the module hasn't been opened, it isopened now. Optionally, the remaining sample fluid is first routed backinto the sample chamber to prevent contamination of the wash reagent:first wash reagent is drawn from reagent chamber A 2530A into one of thesample conduit branches by operating the pump to apply negative pressurewith reagent chamber A vent valve 2432A and the corresponding wastechamber vent valve 2442A or B open (and, preferably, overcoming acapillary break provided by a z-transition in the reagent conduit); thenexcess sample is drawn into the sample chamber by operating the pump toapply positive pressure to the waste chamber vent with the samplechamber vent valve open (FIG. 26, panels 2611-26120. Wash reagent isthen drawn from reagent chamber A 2530A, through detection chambers2550A and 2550B and into waste chambers 2540A and 2540B by operating thepump with reagent chamber A vent valve 2432A and waste chamber ventvalves 2442A and/or 2442B (simultaneously or sequentially) open (FIG.26, panels 2613-1616). As shown, in particularly preferred embodiments,the wash fluid may be segmented, i.e., broken up by one or more slugs ofair. It has been observed that wash fluid alternating with air withinthe detection chambers increases the effectiveness of the clean cycle.Segmenting the wash fluid can be accomplished by periodically andtemporarily opening the air vent valve 2422 and simultaneously closingthe reagent chamber A vent valve 2432A so that air is drawn into thesample conduit. Timing and duration of these operations would dictatethe size and frequency of the air slugs introduced into the segmentedwash fluid slug.

In the two step format, one or more labeled detection reagents may beincubated in the detection chambers in an additional incubation step.Preferably, the detection reagent solution is prepared by reconstitutinga dry reagent pill comprising the detection reagents with an assaydiluent contained within reagent chamber B 2530B. If the assay diluentis in a reagent module (preferably an ampoule) and it is not alreadybroken, it is broken now. The assay diluent is drawn into elongatedreagent conduit 2535 by aspirating at one of the waste chamber ventswhile opening reagent chamber B vent valve 2432B until the assay diluentreaches sensor 13 (FIG. 26, panel 2617). A defined volume of assaydiluent is prepared by closing reagent chamber B vent valve 2432B andopening air vent valve 2422 and continuing to aspirate at the wastechamber vent; reconstitution of the dry reagent in the elongated reagentconduit is promoted by alternating the pump between positive andnegative pressure so as to move the slug back and forth over the dryreagent pill (FIG. 26, panel 2618-2619). In a process analogous to theintroduction of sample to the detection chambers, the slug of detectionreagent solution is i) distributed between the sample conduit branches2515 A and B, ii) introduced to the detection chambers (2550 A and B),incubated in the detection chambers while moving the slugs back in forthin the chambers to increase the rate of the binding of the detectionreagents to immobilized assay components in the chambers, and iii)expelled from the detection chambers to the waste chambers 2540 A and B(FIG. 26, panels 2620-2622). Optionally, residual detection reagentsolution is washed from the detection chambers 2550A and B by aspiratingat the waste chamber vents with the reagent chamber B vent valve 2432Bopen (and, preferably, alternating opening reagent chamber B vent valve2432B and air vent valve 2422 so as to segment the fluid stream) andthen with air vent valve 2422 continuously open to draw the excess assaydiluent into the waste chambers (FIG. 26, panels 2623-2625).Alternatively, washing can be accomplished using the wash reagent byrepeating the steps in panels 2613-2616.

To provide an appropriate environment for the ECL measurement, detectionchambers 2550A and 2550B are filled with the wash reagent (whichpreferably, is an ECL read buffer comprising an ECL coreactant).Accordingly, wash reagent is introduced into the detection chambers byoperating the pump with reagent A chamber vent valve 2432A and wastechamber vent valves 2442A and/or 2442B open so as to aspirate washreagent into sample conduit branches 2515A and 2515B. Operating the pumpwith air vent valve 2422 and waste chamber valves 2442A and/or 2442Bopen introduces slugs wash fluid into the detection chambers (FIG. 16,panels 2628-2631). The above assay is described for a two-step assaythat employs two binding steps. An analogous protocol may be used for aone step protocol with one binding step, preferably, by omitting thesteps in FIG. 26, panels 2617-2625. In the one step format, all thedetection reagents used in the assay are, preferably, stored as dryreagents in sample conduit branches 2515A and 2515B so that they arereconstituted during passage of the sample through the branches.Optionally, reagent chamber B 2530B may be omitted.

Preferably, an ECL measurement is conducted by stimulating/firingworking electrodes in the detection chamber. Preferably, the immobilizedbinding reagents of the detection chambers are immobilized on one ormore working electrodes, more preferably on an array of electrodes, mostpreferably an array of electrodes configured to be fired in a pair-wisefashion (as described above). Electrical potential is applied to theworking electrodes to stimulate ECL, preferably in the pair-wise fashiondiscussed above. The light so generated is detected using an opticaldetector, e.g., using a photodiode or the like. The cartridge and/orlight detector may be moved during the pair-wise firing process so as toalign the active electrode with the light detector. Optionally, an arrayof light detectors or a sufficiently large light detector is used sothat movement of the cartridge and/or light detector is not required.Predefined assay-specific conversion parameters may be used to deriveconcentrations/results from the measured ECL counts; e.g., empiricallyderived from test data or computed from theoretical predictions/models.In particularly preferred embodiments different types of cartridges mayhave different electrode patterns but would preferably employ a commoncartridge electrode contact pattern/area. Some of the electrode contactsmay not be used for lower density cartridge formats.

A preferred sequence of operations that one embodiment of the cartridgereader may employ for firing each read location will now be described.The discussion will reference a photodiode as the optical detector butit should be understood that any suitable optical detector know in theart may be employed. The photodiode assembly (or alternatively, thecartridge) is moved into position; e.g., to the appropriate side of thecartridge's electrode array. The cartridge is then positioned such thatthe first read location to be processed is brought into a predeterminedalignment position with the photodiode (e.g., positioned in registeredalignment) and electrical contact is made to the electrode contacts.Once the contact has been made, the reader preferably performs adiagnostic measurement to detect potential anomalies that may interferewith proper operation of the electrode array and/or its components(leads, contacts, electrodes, etc.). Anomalies that are preferablydetected include manufacturing defects, surface bubbles, or the like.This diagnostic measurement may be accomplished by preferably applyingeither a 500 Hz AC voltage or a very low voltage (e.g., less than 100mV), low current (e.g., less than 1 μA) DC signal to the electrodes andmeasuring the surface capacitance. An appropriate predeterminedalgorithm could then be utilized to determine the presence and/or effectof any such anomalies; e.g., compare measured signal to fixedthresholds, or the like. Preferably, if anomalies are detected, thecartridge reader would record the error and proceed accordingly; e.g.,if the anomaly is isolated to a particular electrode/electrode pair, thecartridge reader would skip reading this location and proceed to thenext pair and/or next operation. Upon confirming operational status, ECLfrom the first pair of electrodes is initiated by application of avoltage waveform; data acquisition from the light detector is alsobegun. After completion of the ECL measurement, the cartridge/lightdetector are realigned to measure ECL from the second electrode pair andthe ECL induction/measurement process is repeated. The cycle is repeatedfor each electrode pair to be analyzed.

In certain preferred embodiments, once a full set of data points hasbeen acquired, the cartridge reader can either store the acquired datalater retrieval/inspection, preferably on machine readable storagemedium, and conclude the read cycle by performing the necessaryfinalization steps (detailed below) or can post-process, preferablyperformed in real-time, the acquired data and store either thepost-processed data alone or in combination with the raw acquired data.Since it is often times important to inspect raw data (e.g.,troubleshooting, diagnostics, data cleansing/filtering, and the like),where data is stored only in post-processed format, the correspondingparameters utilized in converting the data may be stored as well so thatthe raw acquired data can be computed/determined as needed.Alternatively, both the raw acquired data as well as the post-processeddata may be stored. Still further, the raw acquired data may only besubjected to a subset of predetermined data conversion/analysisoperations in real-time and stored for further post-processing offline,i.e., not in real time; post-processing can be performed by thecartridge reader itself or another device, e.g., a general purposeprogrammable computer.

In certain preferred embodiments employing ECL detection technology,data conversion/analysis operations may include one or more of:background subtraction; conversion to ECL counts; conversion of ECLcounts to concentrations; and/or performance of quality checks on theacquired data. Since it is preferable that the resulting data setrepresents only the light generated by ECL background subtraction isemployed to adjust the measured light to correct for the influence ofambient light or “background” signal. Background subtraction consists ofsubtracting the background signal from the photodiode signal.

ECL counts are preferably converted to concentrations usingpredetermined calibration parameters; calibration parameter may bedependent upon one or more factors, e.g., the particular assay/assayformat to be performed within the cartridge, the assay reagentsemployed, the detection technology/techniques employed, cartridgeconfiguration, and the like. Preferably, the calibration parameters areascertained from machine readable indicia associated with the cartridge,e.g., a barcode affixed to or inscribed on the cartridge body. It shouldbe recognized that conversion to ECL counts can occur in a number ofdiffering ways, including, converting all the acquired data points afteracquiring all data, converting each individually acquired data point asit is acquired, converting groups/groupings of acquired data points(e.g., if the cartridge employs a dual read chamber design, convertingto ECL counts upon acquiring the data for each read chamber), etc.

In certain preferred embodiments it is preferable to perform qualitychecks, i.e., assess the quality of the acquired data. Where ECLdetection technology is employed, useful quality checks can be performedon the acquired voltage and current data, including: short circuitdetection; open circuit detection; voltage following confirmation; andpeak current detection. For open and short circuit detection, the outputvoltage and monitored current are preferably integrated for eachacquired data point and the ratio of these two values (current relativeto applied voltage) can then be compared against threshold values; thesethreshold values may be assay-dependent. Results with very low relativecurrent are preferably flagged as probable open circuit conditions whileresults with very high relative current are preferably flagged asprobable short circuits. This information can be stored in relationalform for later review/consideration. Alternatively, if either conditionis detected, the results can be considered invalid and concentrationsfor those measurements not reported/computed.

In the case where a voltage following quality assessment is to beemployed, each point of the acquired voltage waveform is preferablycompared to its corresponding point in a sampled output waveform.Preferably, a predetermined fixed voltage following limit value isdefined for the instrument (i.e., cartridge reader/cartridge) and if anypair of points differs by more than that predetermined value (i.e.,|v(t)_(defined)−v(t)_(measured)|<voltage following limit), the resultsare preferably flagged or considered invalid. If the results areflagged, this information can be stored in relational form for laterreview/consideration. If the results are considered invalid, thecomputed results for those data points are preferably notreported/computed.

Finalization of the cartridge read operation can occur once all of therequisite measurements have been made and all the requisite fluidprocessing has occurred (e.g., once the final measurements have beenmade, route all remaining fluid(s) within the channels and/or readchamber(s) into the waste chamber(s)) the cartridge may be ejected fromthe reader. The cartridge ejection operation preferably occurs inreverse of the operation used to draw the cartridge within the reader.Specifically, the cartridge reader controller ensures that the pump ventis open and that all other valves are closed. Confirmation that the pumpis stopped and all electrode contacts are tri-stated is obtained and, ifa cartridge heater is present and employed, deactivate the cartridgeheater. The cartridge is then preferably moved back onto the reader trayand the reader tray is ejected leaving the cartridge external to thereader and ready for the user, or optionally an automated system, toremove the cartridge from the tray and dispose of it properly.

A preferred embodiment of the performance of an assay using cartridge3200 is described below, the description focusing on aspects that differfrom the operational steps described for cartridge 2500. The operationaldescription includes the use of a preferred valve configuration in thecartridge reader that is similar to that described in FIG. 24 exceptthat it is configured so that air vent port 3244 and air bubble trapvent port 3266 can be connected to the pump, sealed or vented to theatmosphere. In view of the operational description provided forcartridge 2500, the basic operations that are used to move fluid in thispreferred embodiment (i.e., opening vent ports on one side of the fluidto be moved to air and applying positive or negative pressure to a ventport on the other side of the liquid) will be apparent and are notalways described.

A sample, preferably a sample comprising and/or collected on a solidmatrix, is inserted in sample chamber 3220 and cap 3297 is closed. In anespecially preferred embodiment, the sample (most preferably an upperrespiratory sample and/or a sample suspected of containing astreptococcus strain) was collected on an applicator stick (preferably aswab), the applicator stick preferably comprises a pre-defined weakpoint and the sample chamber is curved as shown in FIG. 33. In thisespecially preferred embodiment, insertion of the stick into the curvedchamber causes the shaft to break. The shaft segment is then,preferably, removed and the head segment is sealed in the chamber byclosing cap 3297.

The cartridge is inserted into a reader and mated to the appropriateelectrical and fluidic connections as described above for cartridge2500. The cartridge preferably holds ampoules of extraction and washbuffer in, respectively, reagent chambers 3210 and 3240 which arepreferably broken now (or alternatively any time before they arerequired). The extraction reagent (preferably, nitrous acid, morepreferably, nitrous acid made from a liquid acid in a reagent ampouleand a dry nitrate salt present outside the ampoule in chamber 3210) ispulled from its reagent chamber 3210 by opening vent port 3212 to air,vent port 3244 or 3264 to the pump, and operating the pump to draw theextraction reagent through the swab. To eliminate bubbles in the sample,the pump is operated until fluid from the swab is detected at sensorposition #1. The fluid is then pushed into bubble trap 3226 by openingvent port 3266 to air and operating the pump to apply positive pressureat vent port 3244 or 3264 (or the reverse, i.e., applying negativepressure at vent port 3266 and opening vent port 3244 or 3264 to air).In bubble trap 3226, the bubbles rise to the top of the trap leavingbubble free liquid at the bottom of the trap. More fluid from the swabis pulled up to sensor #1 and again pushed into the bubble trap. This isrepeated as often as necessary to ensure enough bubble-free liquid iscollected in the bubble trap to conduct the assay.

Bubble-free sample liquid is then drawn from the bottom of bubble trap3226 (by aspirating from vent port 3244 or 3264 with vent port 3266 opento air) until the fluid front reaches sensor #1. Vent port 3266 isclosed and vent port 3262 is opened to air and the defined slug ofsample is drawn forward, pulling air behind it from vent port 3262. Thisprocess accurately measures out a defined volume of sample liquid. Thesample slug is then drawn across dry assay reagent 3225 to dissolveit—this reagent preferably includes buffers, labeled binding reagents(preferably antibodies) for the assays, stabilizing reagents, and/orother additives such as blocking reagents. For assays employing nitrousacid as an extraction reagent, the dry assay reagent preferablycomprises sufficient base (preferably, the base form a pH buffer such asTris, Hepes, phosphate, PIPES, etc.) to bring the pH of the sample tobetween 4-10, more preferably between 5-9, more preferably between 6-8.The dissolved reagents may be mixed into the sample by moving the sampleback and forth in the fluid line, using sensors to ensure that theliquid remains within a defined region of conduit.

The sample containing the reconstituted assay reagents is then drawninto detection chamber 3230, where immobilized binding agents(preferably antibodies) are present on individual binding zones thatare, more preferably, located on electrodes in an electrode array. Thesample is incubated for a specific time period over the binding zones,either in a static mode or under mixing, during which time the analyteand labeled binding reagent can bind to each other and/or to theindividual binding zones. Mixing is performed by moving the sampleback-and-forth between sensors at the end of the read chamber.

Sometime before, during, or after sample incubation, a positive controlassay is also performed in the other binding chamber: wash buffer ispulled from the wash buffer storage chamber 3240 to sensor #2 by pullingvacuum on vent port 3264 with vent port 3241 open to air. A fluid slugis metered by closing vent port 3241 and opening vent port 3244 tointroduce air behind the metered fluid as it is drawn toward controldetection chamber 3250. The metered fluid slug is then drawn over anddissolves dry control reagents 3252. These reagents, preferably, includelabeled binding reagents (preferably antibodies), defined amounts of theanalytes for the assays (to provide positive controls), stabilizingreagents and/or other assay reagents. The positive control sample,comprising the metered wash buffer slug and rehydrated control reagents,is then incubated in the control detection chamber 3250 either in astatic fashion or with mixing by moving the sample between sensorslocated at the end of the control binding zone.

Following the incubation steps, the positive control sample is drawninto waste chamber 3254 and the extracted swab sample is drawn into thewaste chamber 3228. Both detection chambers are washed in a consecutiveor simultaneous manner by drawing wash buffer from wash buffer chamber3240 through the detection chambers and into their corresponding wastechambers (waste chamber 3228 for detection chamber 3230 and wastechamber 3254 for control detection chamber 3250). The wash reagent usedduring the wash step is preferably segmented by introducing air at ventport 3244. After washing, both the control and sample binding zones arefilled with wash buffer to complete the fluid sequence. Advantageously,wash reagent flows through detection chamber 3230 in a directionopposite that in which sample was introduced into chamber 3230. Thisreverse flow wash ensures the efficient removal of any components in thesample and/or extraction buffer that could interfere with a measurementin the detection chamber.

Preferably, the binding of analyte and/or labeled binding reagents tobinding domains in the detection chambers is measured by an ECLmeasurement as described above for cartridge 2500. ECL is initiated byapplying the desired electrical potentials to electrodes supporting thebinding zones. The positive control binding zones in detection chamber3250 will provide a positive signal for each assay and may be used toprovide assurance that the assay reagents onboard the cartridge have notdegraded. The ECL signal from any of the sample binding zones indetection chamber 3230 indicates the presence of analyte binds to thatcapture zone or competes with the binding of a labeled reagent to thatcapture zone.

The assay modules (preferably assay cartridges) of the invention may beused to carry out a variety of different assay formats for measuringanalytes interest, preferably formats based on electrode inducedluminescence measurements. The assays, preferably, comprise the steps ofintroducing a sample, and optionally one or more solution phase assayreagents, into an detection chamber (preferably a flow cell) thatcomprises one or more assay domains (preferably a plurality of assaydomains) comprising immobilized assay reagents that bind (with at leastsome degree of selectivity) with analytes of interest. Preferably, thereare at least two assay domains that comprise binding immobilized bindingreagents that differ in their selectivity for analytes. Preferably,there is a patterned array of immobilized binding reagents. Thedetection chamber preferably comprises a plurality of electrodesincluding one or more assay working electrodes having assay domains. Insuch a case, electrical energy is applied to the electrodes (e.g., in apair wise fashion as described above) to induce an assay dependentsignal (e.g., an electrochemical signal such as a current or potentialor, preferably, an electrode induced luminescence signal, mostpreferably an electrochemiluminescence signal) at the electrodes whichis dependent on the amounts of the analytes of interest present in thesample. The assay dependent signal is measured to determine the amountsof the analytes of interest. The assays may comprise the step of washingthe electrodes with a wash solution or they may be carried out in anon-wash format. In washed electrochemiluminescence assays, the assaypreferably comprises the steps of washing the electrodes with a solutioncomprising an electrochemiluminescence coreactant (e.g., a tertiaryalkyl amine such as tripropylamine or PIPES; for other examples ofsuitable coreactants see copending U.S. patent application Ser. No.10/238,437 filed Sep. 10, 2002) and inducing ECL in the presence of thecoreactant. In non-washed ECL assays, a coreactant is preferablyintroduced into the detection chamber with the sample or is present inthe detection chamber prior to the introduction of the sample.Advantageously, assay modules comprising a plurality of assay domains,preferably on a plurality of electrodes, may be used to conduct assaysfor a plurality of analytes of interest.

In preferred embodiments of the invention, the assay modules(preferably, assay cartridges) of the invention are used to carry outbinding assays, most preferably sandwich or competitive binding assays,preferably sandwich or competitive immunoassays. Such assays may,optionally, comprise the step of introducing into the detection chamberlabeled binding reagents such as a labeled binding partner of theanalyte of interest or a labeled competitor that competes with theanalyte of interest for a binding partner of the analyte of interest.Alternatively, these reagents may be stored in dry or wet form in thedetection chamber. For more information on the conduct of bindingassays, particularly using electrochemiluminescence based detection, seecopending U.S. patent application Ser. No. 10/185,274, filed Jun. 28,2002 and copending U.S. patent application Ser. No. 10/238,391, filedSep. 10, 2002, these patent applications hereby incorporated byreference.

The assay modules (preferably, assay cartridges) may be used to carryout panels of assays. Suitable panels include panels of assays foranalytes or activities associated with a specific biochemical system,biochemical pathway, tissue, organism, cell type, organelle, diseasestate, class of receptors, class of enzymes, class of pathogen,environmental sample, food sample, etc. Preferred panels includeimmunoassay for cytokines and/or their receptors (e.g., one or more ofTNF-α, TNF-β, IL-1-α, IL1-β, IL2, IL4, IL6, IL10, IL12, IFN-γ, etc.),growth factors and/or their receptors (e.g., one or more of EGF, VGF,TGF, VEGF, etc.), second messengers (e.g., cAMP, cGMP, phosphorylatedforms of inositol and phosphatidyl inositol, etc.) drugs of abuse,therapeutic drugs, auto-antibodies (e.g., one or more antibodiesdirected against the Sm, RNP, SS-A, SS-B Jo-1, and Scl-70 antigens),allergen specific antibodies, tumor markers, cardiac markers (e.g., oneor more of Troponin T, Troponin I, myoglobin, CKMB, etc.), markersassociated with hemostasis (e.g., one or more of Fibrin monomer,D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 & 2,anti-Factor Xa, etc.), markers of acute viral hepatitis infection (e.g.,one or more of IgM antibody to hepatitis A virus, IgM antibody tohepatitis B core antigen, hepatitis B surface antigen, antibody tohepatitis C virus, etc.), markers of Alzheimers Disease (β-amyloid,tau-protein, etc.), markers of osteoporosis (e.g., one or more ofcross-linked N or C-telopeptides, total deoxypyridinoline, freedeoxypyridinoline, osteocalcin, alkaline phosphatase, C-terminalpropeptide of type I collagen, bone-specific alkaline phosphatase,etc.), markers of fertility (e.g., one or more of Estradiol,progesterone, follicle stimulating hormone (FSH), luetenizing hormone(LH), prolactin, β-hCG, testosterone, etc.), markers of congestive heartfailure (e.g., one or more of β-natriuretic protein (BNP), a-natriureticprotein (ANP), endothelin, aldosterone, etc.), markers of thyroiddisorders (e.g., one or more of thyroid stimulating hormone (TSH), TotalT3, Free T3, Total T4, Free T4, and reverse T3), and markers ofprostrate cancer (e.g., one or more of total PSA, free PSA, complexedPSA, prostatic acid phosphatase, creatine kinase, etc.), pathogensassociated with upper respiratory infection (e.g., influenza A,influenza B, Respiratory Syncytial Virus, Streptococci species),pathogens found in food and water (e.g., salmonella, listeria,cryptosporidia, campylobacter, E. Coli 0157, etc.), sexually transmitteddiseases (e.g., HIV, syphilis, herpes, gonorrhea, HPV, etc.), bloodborne pathogens and potential bioterrorism agents (e.g., pathogens andtoxins in the CDC lists of Select A, B and C agents such as B.anthracis, Y. pestis, small pox, F. tularensis, ricin, botulinum toxins,staph enterotoxins, etc.). Preferred panels also include nucleic acidarrays for measuring mRNA levels of mRNA coding for cytokines, growthfactors, components of the apoptosis pathway, expression of the P450enzymes, expression of tumor related genes, pathogens (e.g., thepathogens listed above), etc. Preferred panels also include nucleic acidarrays for genotyping individuals (e.g., SNP analysis), pathogens, tumorcells, etc. Preferred panels also include libraries of enzymes and/orenzyme substrates (e.g., substrates and/or enzymes associated withubiquitination, protease activity, kinase activity, phosphataseactivity, nucleic acid processing activity, GTPase activity, guaninenucleotide exchange activity, GTPase activating activity, etc.).Preferred panels also include libraries of receptors or ligands (e.g.,panels of G-protein coupled receptors, tyrosine kinase receptors,nuclear hormone receptors, cell adhesion molecules (integrins, VCAM,CD4, CD8), major bistocompatibility complex proteins, nicotinicreceptors, etc.). Preferred panels also include libraries of cells, cellmembranes, membrane fragments, reconstituted membranes, organelles, etc.from different sources (e.g., from different cell types, cell lines,tissues, organisms, activation states, etc.).

The present invention also includes kits. The kits may includedisassembled components necessary to make an assay module of theinvention. Alternatively, the kits may comprise, in one or morecontainers, an assay module of the invention and at least one additionalassay reagent necessary to carry out an assay. The one or more assayreagents may include, but are not limited to, binding reagents(preferably, labeled binding reagents, more preferably binding reagentslabeled with electrochemiluminescent labels) specific for an analyte ofinterest, ECL coreactants, enzymes, enzyme substrates, extractionreagents, assay calibration standards or controls, wash solutions,diluents, buffers, labels (preferably, electrochemiluminescent labels),etc. Preferred kits of the invention include cartridges adapted forextracting samples (as described in detail above), preferably samplescollected on applicator sticks. These kits preferably include applicatorsticks (more preferably swabs) that have properties that are matched tothe specific cartridge. Most preferably, the applicator sticks have weakpoints that are matched to the geometry of a sample introduction chamberin the cartridge such that i) the sticks may be inserted and cleaved inthe cartridge to form a head segment and ii) the head segment can besealed in the sample chamber. Such kits may also include extractionbuffers for extracting the sample on the applicator stick. Oneembodiment of the invention is a ket for measuring upper respiratorypathogens or pathogens that may be found in mucus-containing samples.The kit includes an applicator stick (preferably, a swab) for collectingthe sample (the stick preferably comprising a weak point) and acartridge for measuring a panel of pathogens (e.g., a panel of upperrespiratory pathogens, a panel of sexually transmitted diseases, a panelof pathogens that dwell in mucous membranes, etc.), the cartridgepreferably comprising one or more binding domains containing bindingreagents that bind markers of these pathogens. The kit may also contain(in the cartridge or as a separate component), one or more labeledbinding reagents against markers of these pathogens.

The invention includes assay modules preferably assay cartridges) andmodule readers preferably cartridge readers) as described above. Thesemay be supplied as separate components. The invention also includesassays systems that comprise an assay module (preferably a cartridge)and a module reader (preferably a cartridge reader).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims.

What is claimed is:
 1. An assay cartridge comprising: a cartridge bodyhaving a first side and one or more first side cover layers mated tosaid first side such that said cartridge body and said first side coverlayers define one or more first side fluidic networks therebetween,wherein said assay cartridge includes a sample chamber having a sampleintroduction port with a sealable closure and a sample chamber vent portconnected to said sample chamber; a first waste chamber and a firstwaste chamber vent port connected to said waste chamber; a first reagentchamber, a first reagent chamber vent port connected to said firstreagent chamber, and a first reagent chamber conduit connecting saidfirst reagent chamber with a first sample conduit, a second reagentchamber, a second reagent chamber vent port connected to said secondreagent chamber, and a second reagent chamber conduit connecting saidsecond reagent chamber with a second sample conduit, and a firstdetection chamber connected to said sample chamber by said first sampleconduit and to said first waste chamber by a first waste conduit,wherein said sample and waste chamber vent ports are configured toequilibrate fluid in said sample and waste chambers, respectively, withthe atmosphere.
 2. The assay cartridge of claim 1, said cartridge bodyfurther comprising a second side and one or more second side coverlayers mated to said second side, said cartridge body and said secondside cover layers defining one or more second side fluidic networkstherebetween, wherein said cartridge body has at least one through-holethat provides fluidic communication between said first and second sidefluidic networks.
 3. The assay cartridge of claim 2, wherein at leastone of said fluidic networks is defined, at least in part, by recessesin said cartridge body and/or said cover layers.
 4. The assay cartridgeof claim 3, further comprising a gasket having one or more apertures,said gasket disposed between said cartridge body and at least one ofsaid cover layers and wherein at least one of said fluidic networks isdefined, at least in part, by said apertures.
 5. The assay cartridge ofclaim 1, wherein said first sample conduit includes a capillary break.6. The assay cartridge of claim 5, wherein said capillary breakcomprises a z-transition.
 7. The assay cartridge of claim 6, whereinsaid z-transition comprises, in series, first, second, third, fourth andfifth sample conduit segments, each of said segments connected at anangle to the adjacent segments, said segments oriented so that saidfirst and fifth segments are in a first fluidic network, said thirdsegment is in a second fluidic network, and said second and fourthsegments comprise through-holes through said cartridge body.
 8. Theassay cartridge of claim 1, further comprising a dry reagent in saidfirst sample conduit.
 9. The assay cartridge of claim 8, wherein saiddry reagent comprises a labeled binding reagent, a blocking agent, anECL co-reactant, an extraction buffer neutralization reagent, orcombinations thereof.
 10. The assay cartridge of claim 1, furthercomprising an air vent port connected to said first sample conduit. 11.The assay cartridge of claim 1, wherein said first reagent chambercomprises a first liquid reagent.
 12. The assay cartridge of claim 1,further comprising a first reagent ampoule in said first reagentchamber, said first reagent ampoule containing said first liquidreagent.
 13. The assay cartridge of claim 1, further comprising an airvent port connected to said first reagent chamber conduit.
 14. The assaycartridge of claim 1, further comprising a dry reagent in said firstreagent conduit.
 15. The assay cartridge of claim 14, wherein said dryreagent comprises a labeled binding reagent, a blocking agent, an ECLcoreactant, an extraction buffer neutralization reagent, or combinationsthereof.
 16. The assay cartridge of claim 11, wherein said first liquidreagent is a wash buffer, an extraction buffer, an assay diluent, or anECL read buffer.
 17. The assay cartridge of claim 1, wherein said firstdetection chamber comprises an array of binding reagents.
 18. The assaycartridge of claim 1, wherein said first detection chamber comprises oneor more electrodes having binding reagents immobilized thereon.
 19. Theassay cartridge of claim 1, further comprising a second waste chamber, asecond waste chamber vent port connected to said second waste chamberand a second detection chamber connected to said sample chamber or saidfirst sample conduit by said second sample conduit and to said secondwaste chamber by a second waste conduit.
 20. The assay cartridge ofclaim 1, further comprising a second detection chamber connected to saidsample chamber or said first sample conduit by said second sampleconduit and to said first waste chamber by a second waste conduit. 21.The assay cartridge of claim 1, wherein at least a portion of one wallof said detection chamber is substantially transparent so as to allowoptical monitoring of materials in said detection chamber.
 22. The assaycartridge of claim 1, wherein at least a portion of one of said coverlayers is substantially transparent so as to allow the monitoring offluid flow within said cartridge.
 23. The assay cartridge of claim 1,wherein one of said cover layers has i) a first region comprising apatterned array of immobilized binding reagents, said first regiondefining a surface of said detection chamber and ii) a second regionhaving a dry reagent thereon, said second region defining a surface ofsaid sample conduit.
 24. The assay cartridge of claim 1, wherein saidcartridge has two second side cover layers defining two second sidefluidic networks and a first side bridge cover layer that connects saidtwo second side fluidic networks.
 25. The assay cartridge of claim 24,further comprising dry reagents on said first side bridge cover layer.26. The assay cartridge of claim 4 wherein at least a portion of one ormore of said sample chamber, first waste chamber, first detectionchamber, first sample conduit, and a first waste conduit is formed bysealing one or more of said cover layers to said cartridge body or acomponent thereof.
 27. The assay cartridge of claim 26 wherein thedimensions of said first detection chamber are defined by holes in saidgasket.
 28. The assay cartridge of claim 26 wherein holes in said gasketlayer expose regions of said detection chamber to said first sampleconduit, a reagent conduit, or combinations thereof.